Molecular antigen array

ABSTRACT

The invention provides compositions and processes for the production of ordered and repetitive antigen or antigenic determinant arrays. The compositions of the invention are useful for the production of vaccines for the prevention of infectious diseases, the treatment of allergies and the treatment of cancers. Various embodiments of the invention provide for a core particle that is coated with any desired antigen in a highly ordered and repetitive fashion as the result of specific interactions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit of U.S. provisional applicationNo. 60/202,341, filed May 5, 2000, which is incorporated by reference inits entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is related to the fields of molecular biology,virology, immunology and medicine. The invention provides a compositioncomprising an ordered and repetitive antigen or antigenic determinantarray. The invention also provides a process for producing an antigen orantigenic determinant in an ordered and repetitive array. The orderedand repetitive antigen or antigenic determinant is useful in theproduction of vaccines for the treatment of infectious diseases, thetreatment of allergies and as a pharmaccine to prevent or cure cancerand to generate defined self-specific antibodies and specific immuneresponses of the Th2 type.

2. Background Art

Vaccine development for the prevention of infectious disease has had thegreatest impact on human health of any medical invention. It isestimated that three million deaths per year are prevented worldwide byvaccination (Hillemann, Nature Medicine 4: 507 (1998)). The most commonvaccination strategy, the use of attenuated (i.e., less virulent)pathogens or closely related organisms, was first demonstrated by EdwardJenner in 1796, who vaccinated against smallpox by the administration ofa less dangerous cowpox virus. Although a number of live attenuatedviruses (e.g., measles, mumps, rubella, varicella, adenovirus, polio,influenza) and bacteria (e.g., bacille Calmette-Guerin (BCG) againsttuberculosis) are successfully administered for vaccination, there is arisk for the development of serious complications related to a reversionto virulence and infection by the ‘vaccine’ organism, in particular inimmunocompromised individuals.

The specific design of attenuated viruses is now enabled by recombinantDNA technology (i.e., genetic engineering) through the generation ofdeletion or mutation variants. For example, the administration of anengineered Simian Immunodeficiency Virus (SIV) with a deletion withinthe nef gene was shown to protect macaques from subsequent infectionwith a pathogenic SIV strain (Daniel et al., Science 258: 1938-1941(1992)). However, the progression of acquired immunodeficiency syndrome(AIDS)-like symptoms in animals administered attenuated SIV raisessafety concerns (Baba et al., Science 267: 1820-1825 (1995)).

As an alternative approach, attenuated viruses or bacteria may be usedas carriers for the antigen-encoding genes of a pathogen that isconsidered too unsafe to be administered in an attenuated form (e.g.,Human Immunodeficiency Virus (HIV)). Upon delivery of theantigen-encoding gene to the host, the antigen is synthesized in situ.Vaccinia and related avipox viruses have been used as such carriers forvarious genes in preclinical and clinical studies for a variety ofdiseases (e.g., Shen et al., Science 252: 440 (1991)). One disadvantageof this vaccination strategy is that it does not mimic the virionsurface, because the recombinant protein is expressed on the surface ofthe host cell. Additionally, complications may develop inimmunocompromised individuals, as evidenced by life-threateningdisseminated vaccinia infections (Redfield, N. Eng. J. Med. 316: 673(1998)).

A fourth vaccination approach involves the use of isolated components ofa pathogen, either purified from the pathogen grown in vitro (e.g.,influenza hemagglutinin or neuraminidase) or after heterologousexpression of a single viral protein (e.g., Hepatitis B surfaceantigen). For example, recombinant, mutated toxins (detoxified) are usedfor vaccination against diphtheria, tetanus, cholera and pertussistoxins (Levine et al., New generation vaccines, 2nd edn., Marcel Dekker,Inc., New York 1997), and recombinant proteins of HIV (gp120 andfull-length gp160) were evaluated as a means to induce neutralizingantibodies against HIV with disappointing results (Connor et al, J.Virol. 72: 1552 (1998)). Recently, promising results were obtained withsoluble oligomeric gp 160, that can induce CTL response and elicitantibodies with neutralizing activity against HIV-1 isolates (Van Corttet al., J. Virol. 71: 4319 (1997)). In addition, peptide vaccines may beused in which known B- or T-cell epitopes of an antigen are coupled to acarrier molecule designed to increase the immunogenicity of the epitopeby stimulating T-cell help. However, one significant problem with thisapproach is that it provides a limited immune response to the protein asa whole. Moreover, vaccines have to be individually designed fordifferent MHC haplotypes. The most serious concern for this type ofvaccine is that protective antiviral antibodies recognize complex,three-dimensional structures that cannot be mimicked by peptides.

A more novel vaccination strategy is the use of DNA vaccines (Donnellyet al, Ann. Rev. Immunol. 15: 617 (1997)), which may generate MHC ClassI-restricted CTL responses (without the use of a live vector). This mayprovide broader protection against different strains of a virus bytargeting epitopes from conserved internal proteins pertinent to manystrains of the same virus. Since the antigen is produced with mammalianpost-translational modification, conformation and oligomerization, it ismore likely to be similar or identical to the wild-type protein producedby viral infection than recombinant or chemically modified proteins.However, this distinction may turn out to be a disadvantage for theapplication of bacterial antigens, since non-native post-translationalmodification may result in reduced immunogenicity. In addition, viralsurface proteins are not highly organized in the absence of matrixproteins.

In addition to applications for the prevention of infectious disease,vaccine technology is now being utilized to address immune problemsassociated with allergies. In allergic individuals, antibodies of theIgE isotype are produced in an inappropriate humoral immune responsetowards particular antigens (allergens). The treatment of allergies byallergy immunotherapy requires weekly administration of successivelyincreasing doses of the particular allergen over a period of up to 3-5years. Presumably, ‘blocking’ IgG antibodies are generated thatintercept allergens in nasal or respiratory secretions or in membranesbefore they react with IgE antibodies on mast cells. However, noconstant relationship exists between IgG titers and symptom reliefPresently, this is an extremely time- and cost-consuming process, to beconsidered only for patients with severe symptoms over an extendedperiod each year.

It is well established that the administration of purified proteinsalone is usually not sufficient to elicit a strong immune response;isolated antigen generally must be given together with helper substancescalled adjuvants. Within these adjuvants, the administered antigen isprotected against rapid degradation, and the adjuvant provides anextended release of a low level of antigen.

Unlike isolated proteins, viruses induce prompt and efficient immuneresponses in the absence of any adjuvants both with and without T-cellhelp (Bachmann & Zinkernagel, Ann. Rev. Immunol. 15: 235-270 (1997)).Although viruses often consist of few proteins, they are able to triggermuch stronger immune responses than their isolated components. For Bcell responses, it is known that one crucial factor for theimmunogenicity of viruses is the repetitiveness and order of surfaceepitopes. Many viruses exhibit a quasi-crystalline surface that displaysa regular array of epitopes which efficiently crosslinksepitope-specific immunoglobulins on B cells (Bachmann & Zinkernagel,Immunol. Today 17: 553-558 (1996)). This crosslinking of surfaceimmunoglobulins on B cells is a strong activation signal that directlyinduces cell-cycle progression and the production of IgM antibodies.Further, such triggered B cells are able to activate T helper cells,which in turn induce a switch from IgM to IgG antibody production in Bcells and the generation of long-lived B cell memory—the goal of anyvaccination (Bachmann & Zinkernagel, Ann. Rev. Immunol. 15: 235-270(1997)). Viral structure is even linked to the generation ofanti-antibodies in autoimmune disease and as a part of the naturalresponse to pathogens (see Fehr, T., et al., J. Exp. Med. 185: 1785-1792(1997)). Thus, antigens on viral particles that are organized in anordered and repetitive array are highly immunogenic since they candirectly activate B cells.

In addition to strong B cell responses, viral particles are also able toinduce the generation of a cytotoxic T cell response, another crucialarm of the immune system. These cytotoxic T cells are particularlyimportant for the elimination of non-cytopathic viruses such as HIV orHepatitis B virus and for the eradication of tumors. Cytotoxic T cellsdo not recognize native antigens but rather recognize their degradationproducts in association with MHC class I molecules (Townsend & Bodmer,Ann. Rev. Immunol. 7: 601-624 (1989)). Macrophages and dendritic cellsare able to take up and process exogenous viral particles (but not theirsoluble, isolated components) and present the generated degradationproduct to cytotoxic T cells, leading to their activation andproliferation (Kovacsovics-Bankowski et al., Proc. Natl. Acad. Sci. USA90: 4942-4946 (1993); Bachmann et al., Eur. J. Immunol. 26: 2595-2600(1996)).

Viral particles as antigens exhibit two advantages over their isolatedcomponents: (1) Due to their highly repetitive surface structure, theyare able to directly activate B cells, leading to high antibody titersand long-lasting B cell memory; and (2) Viral particles but not solubleproteins are able to induce a cytotoxic T cell response, even if theviruses are non-infectious and adjuvants are absent.

Several new vaccine strategies exploit the inherent immunogenicity ofviruses. Some of these approaches focus on the particulate nature of thevirus particle; for example see Harding, C. V. and Song, R., (J.Immunology 153: 4925 (1994)), which discloses a vaccine consisting oflatex beads and antigen; Kovacsovics-Bankowski, M., et al. (Proc. Natl.Acad. Sci. USA 90: 4942-4946 (1993)), which discloses a vaccineconsisting of iron oxide beads and antigen; U.S. Pat. No 5,334,394 toKossovsky, N., et al., which discloses core particles coated withantigen, U.S. Pat. No. 5,871,747, which discloses synthetic polymerparticles carrying on the surface one or more proteins covalently bondedthereto; and a core particle with a non-covalently bound coating, whichat least partially covers the surface of said core particle, and atleast one biologically active agent in contact with said coated coreparticle (see, e.g., WO 94/15585).

However, a disadvantage of these viral mimicry systems is that they arenot able to recreate the ordered presentation of antigen found on theviral surface. Antigens coupled to a surface in a random orientation arefound to induce CTL response and no or only weak B-cell response. For anefficient vaccine, both arms of the immune system have to be stronglyactivated, as described above and in Bachmann & Zinkernagel, Ann. Rev.Immunol. 15: 235 (1997).

In another example, recombinant viruses are being utilized for antigendelivery. Filamentous phage virus containing an antigen fused to acapsid protein has been found to be highly immunogenic (see Perham R.N., et al., FEMS Microbiol. Rev. 17: 25-31 (1995); Willis et al., Gene128: 85-88 (1993); Minenkova et al., Gene 128: 85-88 (1993)). However,this system is limited to very small peptides (5 or 6 amino acidresidues) when the fusion protein is expressed at a high level (Iannoloet al., J. Mol Biol. 248: 835-844 (1995)) or limited to the low levelexpression of larger proteins (de la Cruz et al., J. Biol. Chem. 263:4318-4322 (1988)). For small peptides, so far only the CTL response isobserved and no or only weak B-cell response.

In yet another system, recombinant alphaviruses are proposed as a meansof antigen delivery (see U.S. Pat. Nos. 5,766,602; 5,792,462; 5,739,026;5,789,245 and 5,814,482). Problems with the recombinant virus systemsdescribed so far include a low density expression of the heterologousprotein on the viral surface and/or the difficulty of successfully andrepeatedly creating a new and different recombinant viruses fordifferent applications.

In a further development, virus-like particles (VLPs) are beingexploited in the area of vaccine production because of both theirstructural properties and their non-infectious nature. VLPs aresupermolecular structures built in a symmetric manner from many proteinmolecules of one or more types. They lack the viral genome and,therefore, are noninfectious. VLPs can often be produced in largequantities by heterologous expression and can be easily be purified.

Examples of VLPs include the capsid proteins of Hepatitis B virus(Ulrich, et al., Virus Res. 50: 141-182 (1998)), measles virus (Warnes,et al., Gene 160: 173-178 (1995)), Sindbis virus, rotavirus (U.S. Pat.Nos. 5,071,651 and 5,374,426), foot-and-mouth-disease virus (Twomey, etal., Vaccine 13: 1603-1610, (1995)), Norwalk virus (Jiang, X., et al.,Science 250: 1580-1583 (1990);Matsui, S. M., et al., J. Clin. Invest.87: 1456-1461 (1991)), the retroviral GAG protein (PCT Patent Appl. No.WO 96/30523), the retrotransposon Ty protein p1, the surface protein ofHepatitis B virus (WO 92/11291) and human papilloma virus (WO 98/15631).In some instances, recombinant DNA technology may be utilized to fuse aheterologous protein to a VLP protein (Kratz, P. A., et al., Proc. Natl.Acad. Sci. USA 96: 19151920 (1999)).

Thus, there is a need in the art for the development of new and improvedvaccines that promote a strong CTL and B-cell immune response asefficiently as natural pathogens.

BRIEF SUMMARY OF THE INVENTION

The invention provides a versatile new technology that allows productionof particles or pili coated with any desired antigen. The technologyallows the creation of highly efficient vaccines against infectiousdiseases and for the creation of vaccines for the treatment of allergiesand cancers. The invention also provides compositions suited for theinduction of Th type 2 T-helper cells (Th2 cells).

Thus, efficient vaccines for the treatment of chronic diseases inducedor accelerated by a Th1 type immune response, such as arthritis,colitis, diabetes and multiple sclerosis can be produced with thetechnology provided by this invention.

In a first embodiment, the invention provides a novel compositioncomprising (A) a non-natural molecular scaffold and (B) an antigen orantigenic determinant.

The non-natural molecular scaffold comprises, or alternatively consistsof, (i) a core particle selected from the group consisting of (1) a coreparticle of non-natural origin and (2) a core particle of naturalorigin; and (ii) an organizer comprising at least one first attachmentsite, wherein said organizer is connected to said core particle by atleast one covalent bond.

In certain specific embodiments, the core particle naturally contains anorganizer. One example of an embodiment of the invention where theorganizer is naturally occurring is the bacterial pilus or pilinprotein. The antigenic determinant may be linked by a cysteine to anaturally occurring lysine residue of the bacterial pili or pilinprotein.

The antigen or antigenic determinant has at least one second attachmentsite which is selected from the group consisting of (i) an attachmentsite not naturally occurring with said antigen or antigenic determinant,and (ii) an attachment site naturally occurring with said antigen orantigenic determinant.

The invention provides for an ordered and repetitive antigen arraythrough an association of the second attachment site to the firstattachment site by way of at least one non-peptide bond. Thus, theantigen or antigenic determinant and the non-natural molecular scaffoldare brought together through this association of the first and thesecond attachment site to form an ordered and repetitive antigen array.

In another embodiment, the core particle of the aforementionedcomposition comprises a virus, a virus-like particle, a bacterial pilus,a structure formed from bacterial pilin, a bacteriophage, a viral capsidparticle or a recombinant form thereof Alternatively, the core particlemay be a synthetic polymer or a metal.

In yet another embodiment, the core particle comprises, or alternativelyconsists of, one or more different Hepatitis core (capsid) proteins(HBcAgs). In a related embodiment, one or more cysteine residues ofthese HBcAgs are either deleted or substituted with another amino acidresidue (e.g., a serine residue). In a specific embodiment, the cysteineresidues of the HBcAg used to prepare compositions of the inventionwhich correspond to amino acid residues 48 and 107 in SEQ ID NO:134 areeither deleted or substituted with another amino acid residue (e.g., aserine residue).

Further, the HBcAg variants used to prepare compositions of theinvention will generally be variants which retain the ability toassociate with other HBcAgs to form dimeric or multimeric structuresthat present ordered and repetitive antigen or antigenic determinantarrays.

In another embodiment, the non-natural molecular scaffold comprises, oralternatively consists of, pili or pilus-like structures that have beeneither produced from pilin proteins or harvested from bacteria. Whenpili or pilus-like structures are used to prepare compositions of theinvention, they may be formed from products of pilin genes which arenaturally resident in the bacterial cells but have been modified bygenetically engineered (e.g., by homologous recombination) or pilingenes which have been introduced into these cells.

In a related embodiment, the core particle comprises, or alternativelyconsists of, pili or pilus-like structures that have been eitherprepared from pilin proteins or harvested from bacteria. These coreparticles may be formed from products of pilin genes naturally residentin the bacterial cells. Further, antigens or antigenic determinants maybe linked to these core particles naturally containing an organizer. Insuch a case, the core particles will generally be linked to a secondattachment site of the antigen or antigenic determinant. In mostembodiments of the invention, the pili or pilus-like structures will beable to form an ordered and repetitive antigen array with the antigen orantigenic determinant linked to the core particle at a specific orpreferred location (e.g., a specific amino acid residue).

In a particular embodiment, the organizer may comprise at least onefirst attachment site. The first and the second attachment sites areparticularly important elements of compositions of the invention. Invarious embodiments of the invention, the first and/or the secondattachment site may be an antigen and an antibody or antibody fragmentthereto; biotin and avidin; strepavidin and biotin;

a receptor and its ligand; a ligand-binding protein and its ligand;interacting leucine zipper polypeptides; an amino group and a chemicalgroup reactive thereto; a carboxyl group and a chemical group reactivethereto; a sulfhydryl group and a chemical group reactive thereto; or acombination thereof

In one embodiment, the invention provides the coupling of almost anyantigen of choice to the surface of a virus, bacterial pilus, structureformed from bacterial pilin, bacteriophage, virus-like particle or viralcapsid particle. By bringing an antigen into a quasi-crystalline‘virus-like’ structure, the invention exploits the strong antiviralimmune reaction of a host for the production of a highly efficientimmune response, i.e., a vaccination, against the displayed antigen.

In another embodiment, the core particle may be selected from the groupconsisting of: recombinant proteins of Rotavirus, recombinant proteinsof Norwalk virus, recombinant proteins of Alphavirus, recombinantproteins of Foot and Mouth Disease virus, recombinant proteins ofRetrovirus, recombinant proteins of Hepatitis B virus, recombinantproteins of Tobacco mosaic virus, recombinant proteins of Flock HouseVirus, and recombinant proteins of human Papilomavirus.

In yet another embodiment, the antigen may be selected from the groupconsisting of: (1) a protein suited to induce an immune response againstcancer cells; (2) a protein suited to induce an immune response againstinfectious diseases; (3) a protein suited to induce an immune responseagainst allergens; and (4) a protein suited to induce an immune responsein pets or farm animals.

In one embodiment, the invention relates to the induction of specific Thtype 2 T-helper cells (Th2 cells) using antigens attached to Pili. Theinduction of Th2 responses may be beneficial for the treatment of anumber of diseases. For example, many chronic diseases in humans ananimals, such as arthritis, colitis, diabetes and multiple sclerosis aredominated by Th1 response, where T cells secrete IFN_(γ) and otherpro-inflammatory cytokines precipitating disease.

In a particularly embodiment of the invention, the first attachment siteand/or the second attachment site comprise an interacting leucine zipperpolypeptide. In a related embodiment, the first attachment site and/orthe second attachment site are selected from the group comprising: (1)the JUN leucine zipper protein domain; and (2) the FOS leucine zipperprotein domain.

In another embodiment, the first attachment site and/or the secondattachment site are selected from the group comprising: (1) agenetically engineered lysine residue and (2) a genetically engineeredcysteine residue, two residues that may be chemically linked together.

The invention also includes embodiments where the organizer particle hasonly a single first attachment site and the antigen or antigenicdeterminant has only a single second attachment site. Thus, when anordered and repetitive antigen array is prepared using such embodiments,each organizer will be bound to a single antigen or antigenicdeterminant.

In one aspect, the invention provides compositions comprising, oralternatively consisting of, (a) a non-natural molecular scaffoldcomprising (i) a core particle selected from the group consisting of acore particle of non-natural origin and a core particle of naturalorigin, and (ii) an organizer comprising at least one first attachmentsite, wherein the core particle comprises, or alternatively consists of,a bacterial pilus, a pilus-like structure, or a modified HBcAg, orfragment thereof, and wherein the organizer is connected to the coreparticle by at least one covalent bond, and (b) an antigen or antigenicdeterminant with at least one second attachment site, the secondattachment site being selected from the group consisting of (i) anattachment site not naturally occurring with the antigen or antigenicdeterminant and (ii) an attachment site naturally occurring with theantigen or antigenic determinant, wherein the second attachment site iscapable of association through at least one non-peptide bond to thefirst attachment site, and wherein the antigen or antigenic determinantand the scaffold interact through the association to form an ordered andrepetitive antigen array.

Other embodiments of the invention include processes for the productionof compositions of the invention and a methods of medical treatmentusing vaccine compositions described herein.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are intended to provide further explanation of the invention asclaimed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a Western blot demonstrating the production of viralparticles containing the E2-JUN fusion protein using the pCYTts::E2JUNexpression vector.

FIG. 2 shows a Western blot demonstrating the production of viralparticles containing the E2-JUN fusion protein expressed frompTE5′2J::E2JUN expression vector.

FIG. 3 shows a Western dot blot demonstrating bacterial and eukaryoticexpression of the FOS-hgh antigen.

FIG. 4 shows the expression of HBcAg-JUN in E. coli cells.

FIG. 5 shows a Western blot demonstrating that HBcAg-JUN is soluble inE. coli lysates.

FIG. 6 shows an SDS-PAGE analysis of enrichment of HBcAg-JUN capsidparticles on a sucrose density gradient.

FIG. 7 shows a non-reducing SDS-PAGE analysis of the coupling of hGH-FOSand HBcAg-JUN particles.

FIG. 8 depicts an analysis by SDS-PAGE of the coupling reaction of theFLAG peptide to HBcAG-Lys treated with iodacetamide and activated withSulfo-MBS. The excess of cross-linker and of peptide over HBcAg-Lysmonomer is indicated below the figure.

FIG. 9 depicts an analysis of coupling of the FLAG peptide to type-1bacterial pili by SD S-PAGE. Lane 1 shows the unreacted pili subunitFimA. Lane 3 shows the purified reaction mixture of the pill with theFLAG peptide. The upper band corresponds to the coupled product, whilethe lower band corresponds to the unreached subunit.

FIG. 10 depicts an analysis by SDS-PAGE of the derivatization ofHBcAg-Lys with SPDP.

FIG. 11 depicts an analysis by SDS-PAGE of the derivatization ofHBcAg-Lys with Sulfo-MBS.

FIG. 12 depicts an analysis by SDS-PAGE of the coupling ofHBcAg-Lys-2cyc-Mut to the FLAG peptide. The arrow shows the bandscorresponding to the coupling of one and two FLAG peptides,respectively, to one subunit of HBcAgLys-2cyc-Mut. Lane M corresponds tothe marker, lane 1 to the unreached HBcAg-Lys-2cyc-Mut, lane 2 toHBcAg-Lys-2cyc-Mut activated with Sulfo-MBS, and lane 3 activatedHBcAg-Lys-2cyc-Mut after reaction with the FLAG peptide containing anN-terminal cysteine.

FIG. 13 depicts an analysis by SDS-PAGE of the coupling of pili to thep33 peptide.

FIG. 14A shows an analysis of coupling of DP178c peptide by SDS-PAGEanalysis and Coomassie staining. Lane 1 corresponds to the supernatantof the coupling reaction after centrifugation, while lane 2 correspondsto the pellet.

FIG. 14B show an ELISA data and subtype analysis of mice, sera immunizedwith Pili-DP178c. The OD (450 nm) of the ELISA signal obtained at afifty-fold dilution of the sera is shown in the diagram. For eachsubtype determination, mice sera were titrated from a fifty-folddilution in two-fold dilution steps. The ELISA titer of the IgG1 subtype(OD50 dilution) was 1:400, while the titer of the IgG2b subtype was1:100. The other subtypes all had titers inferior to 1:50. The IgGisotype pattern is characteristic of a Th2 response, with a high IgG1titer and a low IgG2a titer.

FIG. 15A shows an analysis of Coupling of GRA2 to Pili by SDS-PAGEanalysis and Coomassie staining. FIG. 15B relates to immunization ofmice with Pili-GRA2 and IgG subtype determination. Depicted is ananalysis of total IgG titer and IgG subtype titers by ELISA. The ELISAtiter is given by the dilution of sera at which OD50 is obtained. Theresult of the immunization of two individual mice is shown. A high IgG1titer and a low IgG2a titer is characteristic of a Th2 response.

FIG. 16A shows an analysis of coupling of B2 and D2 peptides to Pili bySDS-PAGE analysis and Coomassie staining. FIG. 16B relates toimmunization of mice with Pili-B2 and IgG subtype determination. The OD(450 nm) of the ELISA signal obtained at a fifty-fold dilution of thesera is shown in the diagram. For each subtype determination, mice serawere titrated from a fifty-fold dilution in two-fold dilution steps. Thetiter of the IgG1 subtype (dilution at which the signal corresponds toOD 50) was 1:250, while the other subtypes all had titers inferior to1:50. The titer of the IgG1 subtype is much higher than the titer of theIgG2a subtype, a pattern typical for a Th2 response.

FIG. 17 relates to the measurement of antibodies specific for TNFαprotein in the serum of mice immunized with the muTNFα peptide coupledto type-1 Pili. As a control, preimmune sera of two mice were assayedfor binding to TNFα protein. Sera were added at three differentdilutions (1:50, 1:100 and 1:200), and bound IgG was detected with ahorseradish peroxidase-conjugated anti-murine IgG antibody. Results fromfour individual mice are shown on day 21 and day 43. OD (450 nm):optical density at 450 nm.

FIG. 18A shows an analysis of coupling of 5′-TNF II and 3′-TNF II bySDS-PAGE and Coomassie staining. Lane M is the marker lane. UntreatedPili were loaded on lane 1, Pili-5′-TNF II before dialysis on lane 2,Pili-3′-TNF II before dialysis on lane 3, Pili-5′-TNF II after dialysison lane 4, pili-3′-TNF II after dialysis on lane 5. The arrow indicatesthe size at which the coupled product migrates.

FIG. 18B shows an ELISA analysis of sera of mice immunized withPili-5′-TNF II and Pili-3′-TNF II: Anti-TNFα ELISA. IgG antibodiesspecific for native TNFα protein were measured in a specific ELISA. 2μg/ml native TNFα protein was coated on ELISA plates. Sera were added atdifferent dilutions and bound IgG was detected with a horseradishperoxidase-conjugated anti-murine IgG antibody. Results from fourindividual mice are shown on day 21 and day 43 OD (450 nm): opticaldensity at 450 nm. The data show that mice immunized with the TNFpeptides coupled to pili mount an antibody response against native TNFαprotein, thus breaking self-tolerance.

FIG. 18C shows an ELISA analysis of sera of mice immunized withPili-5′-TNF II and Pili-3′-TNF II: Anti-TNFα peptide ELISA. IgGantibodies specific for the 5′TNF II and 3′TNF II peptides were measuredin a specific ELISA: 10 μg/ml Ribonuclease A coupled to 5′TNF II or3′TNF II peptide was coated on ELISA plates. Sera were added atdifferent dilutions and bound IgG was detected with a horseradishperoxidase conjugated anti-murine IgG antibody. Results from fourindividual mice are shown on day 21.

FIG. 18D shows that IgG subtype analysis of anti-TNF peptide antibodiesin mice vaccinated with the corresponding TNF-peptides coupled to Pili.Results from four individual mice (no. 1-4) are shown on day 50. ELISAtiter: dilution step at which half-maximal optical density was reached(−log 2 of 40-fold prediluted sera). The high IgG1 titer obtained ascompared to the very low IgG2a titer is typical of a Th2 response.

FIG. 19A shows an analysis of coupling of M2 peptide to Pili by SDS-PAGEanalysis and Coomassie staining. The bands corresponding to non-coupledPili and to the coupling product, Pili-M2, are indicated by arrows. FIG.19B shows an ELISA analysis and IgG subtype determination of micevaccinated with Pili-M2. Sera were diluted eighty-fold, and titrateddown in two-fold dilution steps. For the IgG1 subtype, a titer of 1:2560was obtained, while for the IgG2a and IgG2b subtypes, titers below 1:100were obtained. The titer for the IgG3 subtype was below 1:80. Titerswere calculated as the serum dilution resulting in half-maximal opticaldensity (OD₅₀). A strong IgG1 titer in conjunction with a low IgG2atiter is characteristic for a Th2 type response. Average results fromtwo mice are shown as optical densities obtained with a 1:80 dilution ofthe serum.

FIG. 20 shows an ELISA analysis and IgG subtype determination of serafrom mice immunized with HBcAg-Lys-2cys-Mut coupled to the Flag peptide.Ribonuclease A coupled to Flag peptide was coated at 10 μg/ml, and serumwas added at a 1:40 dilution. In contrast to experiments where mice wereimmunized with antigens coupled to Pili, there is no predominance of theIgG1 subtype over the other IgG subtypes.

DETAILED DESCRIPTION OF THE INVENTION 1. DEFINITIONS

The following definitions are provided to clarify the subject matterwhich the inventors consider to be the present invention.

Alphavirus: As used herein, the term “alphavirus” refers to any of theRNA viruses included within the genus Alphavirus. Descriptions of themembers of this genus are contained in Strauss and Strauss, Microbiol.Rev., 58: 491-562 (1994). Examples of alphaviruses include Aura virus,Bebaru virus, Cabassou virus, Chikungunya virus, Easter equineencephalomyelitis virus, Fort morgan virus, Getah virus, Kyzylagachvirus, Mayoaro virus, Middleburg virus, Mucambo virus, Ndumu virus,Pixuna virus, Tonate virus, Triniti virus, Una virus, Western equineencephalomyelitis virus, Whataroa virus, Sindbis virus (SIN), Semlikiforest virus (SFV), Venezuelan equine encephalomyelitis virus (VEE), andRoss River virus.

Antigen: As used herein, the term “antigen” is a molecule capable ofbeing bound by an antibody. An antigen is additionally capable ofinducing a humoral immune response and/or cellular immune responseleading to the production of B- and/or T-lymphocytes. An antigen mayhave one or more epitopes (B- and T-epitopes). The specific reactionreferred to above is meant to indicate that the antigen will react, in ahighly selective manner, with its corresponding antibody and not withthe multitude of other antibodies which may be evoked by other antigens.

Antigenic determinant: As used herein, the term“antigenic determinant”is meant to refer to that portion of an antigen that is specificallyrecognized by either B- or T-lymphocytes. B-lymphocytes respond toforeign antigenic determinants via antibody production, whereasT-lymphocytes are the mediator of cellular immunity. Thus, antigenicdeterminants or epitopes are those parts of an antigen that arerecognized by antibodies, or in the context of an MHC, by T-cellreceptors.

Association: As used herein, the term “association” as it applies to thefirst and second attachment sites, is used to refer to at least onenon-peptide bond. The nature of the association may be covalent, ionic,hydrophobic, polar or any combination thereof

Attachment Site, First: As used herein, the phrase “first attachmentsite” refers to an element of the “organizer”, itself bound to the coreparticle in a non-random fashion, to which the second attachment sitelocated on the antigen or antigenic determinant may associate. The firstattachment site may be a protein, a polypeptide, an amino acid, apeptide, a sugar, a polynucleotide, a natural or synthetic polymer, asecondary metabolite or compound (biotin, fluorescein, retinol,digoxigenin, metal ions, phenylmethylsulfonylfluoride), or a combinationthereof, or a chemically reactive group thereof Multiple firstattachment sites are present on the surface of the non-natural molecularscaffold in a repetitive configuration.

Attachment Site, Second: As used herein, the phrase “second attachmentsite” refers to an element associated with the antigen or antigenicdeterminant to which the first attachment site of the “organizer”located on the surface of the non-natural molecular scaffold mayassociate. The second attachment site of the antigen or antigenicdeterminant may be a protein, a polypeptide, a peptide, a sugar, apolynucleotide, a natural or synthetic polymer, a secondary metaboliteor compound (biotin, fluorescein, retinol, digoxigenin, metal ions,phenylmethylsulfonylfluoride), or a combination thereof, or a chemicallyreactive group thereof At least one second attachment site is present onthe antigen or antigenic determinant.

Core particle: As used herein, the term “core particle” refers to arigid structure with an inherent repetitive organization that provides afoundation for attachment of an “organizer”. A core particle as usedherein may be the product of a synthetic process or the product of abiological process.

In certain embodiments of the invention, the antigens or antigenicdeterminants are directly linked to the core particle.

Cis-acting: As used herein, the phrase “cis-acting” sequence refers tonucleic acid sequences to which a replicase binds to catalyze theRNA-dependent replication of RNA molecules. These replication eventsresult in the replication of the full-length and partial RNA moleculesand, thus, the alpahvirus subgenomic promoter is also a “cis-acting”sequence. Cis-acting sequences may be located at or near the 5′ end, 3′end, or both ends of a nucleic acid molecule, as well as internally.

Fusion: As used herein, the term “fusion” refers to the combination ofamino acid sequences of different origin in one polypeptide chain byin-frame combination of their coding nucleotide sequences. The term“fusion” explicitly encompasses internal fusions, i.e., insertion ofsequences of different origin within a polypeptide chain, in addition tofusion to one of its termini.

Heterologous sequence: As used herein, the term “heterologous sequence”refers to a second nucleotide sequence present in a vector of theinvention. The term “heterologous sequence” also refers to any aminoacid or RNA sequence encoded by a heterologous DNA sequence contained ina vector of the invention. Heterologous nucleotide sequences can encodeproteins or RNA molecules normally expressed in the cell type in whichthey are present or molecules not normally expressed therein (e.g.,Sindbis structural proteins).

Isolated: As used herein, when the term “isolated” is used in referenceto a molecule, the term means that the molecule has been removed fromits native environment. For example, a polynucleotide or a polypeptidenaturally present in a living animal is not “isolated,” but the samepolynucleotide or polypeptide. separated from the coexisting materialsof its natural state is “isolated.” Further, recombinant DNA moleculescontained in a vector are considered isolated for the purposes of thepresent invention. Isolated RNA molecules include in vivo or in vitroRNA replication products of DNA and RNA molecules. Isolated nucleic acidmolecules further include synthetically produced molecules. Additionallyvector molecules contained in recombinant host cells are also isolated.Thus, not all “isolated” molecules need be “purified.”

Immunotherapeutic: As used herein, the term “immunotherapeutic” is acomposition for the treatment of diseases or disorders. Morespecifically, the term is used to refer to a method of treatment forallergies or a method of treatment for cancer.

Individual: As used herein, the term “individual” refers tomulticellular organisms and includes both plants and animals. Preferredmulticellular organisms are animals, more preferred are vertebrates,even more preferred are mammals and most preferred are humans.

Low or undetectable: As used herein, the phrase “low or undetectable,”when used in reference to gene expression level, refers to a level ofexpression which is either significantly lower than that seen when thegene is maximally induced (e.g., at least five fold lower) or is notreadily detectable by the methods used in the following examplessection.

Lectin: As used herein, proteins obtained particularly from the seeds ofleguminous plants, but also from many other plant and animal sources,that have binding sites for specific mono- or oligosaccharides. Examplesinclude concanavalin A and wheat-germ agglutinin, which are widely usedas analytical and preparative agents in the study of glycoprotein.

Natural origin: As used herein, the term “natural origin” means that thewhole or parts thereof are not synthetic and exist or are produced innature.

Non-natural: As used herein, the term generally means not from naturemore specifically, the term means from the hand of man.

Non-natural origin: As used herein, the term “non-natural origin”generally means synthetic or not from nature; more specifically, theterm means from the hand of man.

Non-natural molecular scaffold: As used herein, the phrase “non-naturalmolecular scaffold” refers to any product made by the hand of man thatmay serve to provide a rigid and repetitive array of first attachmentsites. Ideally but not necessarily, these first attachment sites are ina geometric order. The non-natural molecular scaffold may be organic ornon-organic and may be synthesized chemically or through a biologicalprocess, in part or in whole. The non-natural molecular scaffold iscomprised of: (a) a core particle, either of natural or non-naturalorigin; and (b) an organizer, which itself comprises at least one firstattachment site and is connected to a core particle by at least onecovalent bond.

In a particular embodiment, the non-natural molecular scaffold may be avirus, virus-like particle, a bacterial pilus, a virus capsid particle,a phage, a recombinant form thereof, or synthetic particle.

Ordered and repetitive antigen or antigenic determinant array: As usedherein, the term “ordered and repetitive antigen or antigenicdeterminant array” generally refers to a repeating pattern of antigen orantigenic determinant, characterized by a uniform spacial arrangement ofthe antigens or antigenic determinants with respect to the non-naturalmolecular scaffold. In one embodiment of the invention, the repeatingpattern may be a geometric pattern. Examples of suitable ordered andrepetitive antigen or antigenic determinant arrays are those whichpossess strictly repetitive paracrystalline orders of antigens orantigenic determinants with spacings of 5 to 15 nanometers.

Organizer: As used herein, the term “organizer” is used to refer to anelement bound to a core particle in a non-random fashion that provides anucleation site for creating an ordered and repetitive antigen array. Anorganizer is any element comprising at least one first attachment sitethat is bound to a core particle by at least one covalent bond. Anorganizer may be a protein, a polypeptide, a peptide, an amino acid(i.e., a residue of a protein, a polypeptide or peptide), a sugar, apolynucleotide, a natural or synthetic polymer, a secondary metaboliteor compound (biotin, fluorescein, retinol, digoxigenin, metal ions,phenylmethylsulfonylfluoride), or a combination thereof, or a chemicallyreactive group thereof.

Permissive temperature: As used herein, the phrase “permissivetemperature” refers to temperatures at which an enzyme has relativelyhigh levels of catalytic activity.

Pili: As used herein, the term “pili” (singular being “pilus”) refers toextracellular structures of bacterial cells composed of protein monomers(e.g., pilin monomers) which are organized into ordered and repetitivepatterns. Further, pili are structures which are involved in processessuch as the attachment of bacterial cells to host cell surfacereceptors, inter-cellular genetic exchanges, and cell-cell recognition.Examples of pili include Type-1 pili, P-pili, F1C pili, S-pili, and987P-pili. Additional examples of pili are set out below.

Pilus-like structure: As used herein, the phrase “pilus-like structure”refers to structures having characteristics similar to that of pili andcomposed of protein monomers. One example of a “pilus-like structure” isa structure formed by a bacterial cell which expresses modified pilinproteins that do not form ordered and repetitive arrays that areessentially identical to those of natural pili.

Purified: As used herein, when the term “purified” is used in referenceto a molecule, it means that the concentration of the molecule beingpurified has been increased relative to molecules associated with it inits natural environment. Naturally associated molecules includeproteins, nucleic acids, lipids and sugars but generally do not includewater, buffers, and reagents added to maintain the integrity orfacilitate the purification of the molecule being purified. For example,even if mRNA is diluted with an aqueous solvent during oligo dT columnchromatography, mRNA molecules are purified by this chromatography ifnaturally associated nucleic acids and other biological molecules do notbind to the column and are separated from the subject mRNA molecules.

Receptor: As used herein, the term “receptor” refers to proteins orglycoproteins or fragments thereof capable of interacting with anothermolecule, called the ligand. The ligand may belong to any class ofbiochemical or chemical compounds. The receptor need not necessarily bea membrane-bound protein. Soluble protein, like e.g., maltose bindingprotein or retinol binding protein are receptors as well.

Residue: As used herein, the term “residue” is meant to mean a specificamino acid in a polypeptide backbone or side chain.

Temperature-sensitive: As used herein, the phrase“temperature-sensitive” refers to an enzyme which readily catalyzes areaction at one temperature but catalyzes the same reaction slowly ornot at all at another temperature. An example of a temperature-sensitiveenzyme is the replicase protein encoded by the pCYTts vector, which hasreadily detectable replicase activity at temperatures below 34° C. andhas low or undetectable activity at 37° C.

Transcription: As used herein, the term “transcription” refers to theproduction of RNA molecules from DNA templates catalyzed by RNApolymerase.

Recombinant host cell: As used herein, the term “recombinant host cell”refers to a host cell into which one ore more nucleic acid molecules ofthe invention have been introduced.

Recombinant virus: As used herein, the phrase “recombinant virus” refersto a virus that is genetically modified by the hand of man. The phrasecovers any virus known in the art. More specifically, the phrase refersto a an alphavirus genetically modified by the hand of man, and mostspecifically, the phrase refers to a Sinbis virus genetically modifiedby the hand of man.

Restrictive temperature: As used herein, the phrase “restrictivetemperature” refers to temperatures at which an enzyme has low orundetectable levels of catalytic activity. Both “hot” and “cold”sensitive mutants are known and, thus, a restrictive temperature may behigher or lower than a permissive temperature.

RNA-dependent RNA replication event: As used herein, the phrase“RNA-dependent RNA replication event” refers to processes which resultin the formation of an RNA molecule using an RNA molecule as a template.

RNA-Dependent RNA polymerase: As used herein, the phrase “RNA-DependentRNA polymerase” refers to a polymerase which catalyzes the production ofan RNA molecule from another RNA molecule. This term is used hereinsynonymously with the term “replicase.”

Untranslated RNA: As used herein, the phrase “untranslated RNA” refersto an RNA sequence or molecule which does not encode an open readingframe or encodes an open reading frame, or portion thereof, but in aformat in which an amino acid sequence will not be produced (e.g., noinitiation codon is present). Examples of such molecules are tRNAmolecules, rRNA molecules, and ribozymes.

Vector: As used herein, the term “vector” refers to an agent (e.g., aplasmid or virus) used to transmit genetic material to a host cell. Avector may be composed of either DNA or RNA.

one, a, or an: When the terms “one,” “a,” or “an” are used in thisdisclosure, they mean “at least one” or “one or more,” unless otherwiseindicated.

2. COMPOSITIONS OF ORDERED AND REPETITIVE ANTIGEN OR ANTIGENICDETERMINANT ARRAYS AND METHODS TO MAKE THE SAME

The disclosed invention provides compositions comprising an ordered andrepetitive antigen or antigenic determinant. Furthermore, the inventionconveniently enables the practitioner to construct ordered andrepetitive antigen or antigenic determinant arrays for various treatmentpurposes, which includes the prevention of infectious diseases, thetreatment of allergies and the treatment of cancers. The invention alsoenables the practitioner to construct compositions comprising Piliinducing Th2 immune responses, useful in the treatment of chronicdiseases.

Compositions of the invention essentially comprise, or alternativelyconsist of, two elements: (1) a non-natural molecular scaffold, and (2)an antigen or antigenic determinant with at least one second attachmentsite capable of association through at least one non-peptide bond tosaid first attachment site.

The non-natural molecular scaffold comprises, or alternatively consistsof: (a) a core particle selected from the group consisting of (1) a coreparticle of non-natural origin and (2) a core particle of naturalorigin; and (b) an organizer comprising at least one first attachmentsite, wherein said organizer is connected to said core particle by atleast one covalent bond.

Compositions of the invention also comprise, or alternatively consistof, core particles to which antigens or antigenic determinants aredirectly linked.

The antigen or antigenic determinant has at least one second attachmentsite which is selected from the group consisting of (a) an attachmentsite not naturally occurring with said antigen or antigenic determinant;and (b) an attachment site naturally occurring with said antigen orantigenic determinant.

The invention provides for an ordered and repetitive antigen arraythrough an association of the second attachment site to the firstattachment site by way of at least one non-peptide bond. Thus, theantigen or antigenic determinant and the non-natural molecular scaffoldare brought together through this association of the first and thesecond attachment site to form an ordered and repetitive antigen array.

The practioner may specifically design the antigen or antigenicdeterminant and the second attachment site such that the arrangement ofall the antigens or antigenic determinants bound to the non-naturalmolecular scaffold or, in certain embodiments, the core particle will beuniform. For example, one may place a single second attachment site onthe antigen or antigenic determinant at the carboxyl or amino terminus,thereby ensuring through design that all antigen or antigenicdeterminant molecules that are attached to the non-natural molecularscaffold are positioned in a uniform way. Thus, the invention provides aconvenient means of placing any antigen or antigenic determinant onto anon-natural molecular scaffold in a defined order and in a manner whichforms a repetitive pattern.

As will be clear to those skilled in the art, certain embodiments of theinvention involve the use of recombinant nucleic acid technologies suchas cloning, polymerase chain reaction, the purification of DNA and RNA,the expression of recombinant proteins in prokaryotic and eukaryoticcells, etc. Such methodologies are well known to those skilled in theart and may be conveniently found in published laboratory methodsmanuals (e.g., Sambrook, J. et al., eds., MOLECULAR CLONING, ALABORATORY MANUAL, 2nd. edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1989); Ausubel, F. et al., eds., CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, John H. Wiley & Sons, Inc. (1997)).Fundamental laboratory techniques for working with tissue culture celllines (Celis, J., ed., CELL BIOLOGY, Academic Press, 2^(nd) edition,(1998)) and antibody-based technologies (Harlow, E. and Lane, D.,“Antibodies: A Laboratory Manual,” Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1988); Deutscher, M. P., “Guide to ProteinPurification,” Meth. Enzymol. 128, Academic Press San Diego (1990);Scopes, R. K., “Protein Purification Principles and Practice,” 3^(rd)ed., Springer-Verlag, New York (1994)) are also adequately described inthe literature, all of which are incorporated herein by reference.

A. Construction of a Non-Natural Molecular Scaffold

One element in compositions of the invention is a non-natural molecularscaffold comprising, or alternatively consisting of, a core particle andan organizer. As used herein, the phrase “non-natural molecularscaffold” refers to any product made by the hand of man that may serveto provide a rigid and repetitive array of first attachment sites. Morespecifically, the non-natural molecular scaffold comprises, oralternatively consists of, (a) a core particle selected from the groupconsisting of (1) a core particle of non-natural origin and (2) a coreparticle of natural origin; and (b) an organizer comprising at least onefirst attachment site, wherein said organizer is connected to said coreparticle by at least one covalent bond.

As will be readily apparent to those skilled in the art, the coreparticle of the non-natural molecular scaffold of the invention is notlimited to any specific form. The core particle may be organic ornon-organic and may be synthesized chemically or through a biologicalprocess.

In one embodiment, a non-natural core particle may be a syntheticpolymer, a lipid micelle or a metal. Such core particles are known inthe art, providing a basis from which to build the novel non-naturalmolecular scaffold of the invention. By way of example, syntheticpolymer or metal core particles are described in U.S. Pat. No.5,770,380, which discloses the use of a calixarene organic scaffold towhich is attached a plurality of peptide loops in the creation of an‘antibody mimic’, and U.S. Pat. No. 5,334,394 describes nanocrystallineparticles used as a viral decoy that are composed of a wide variety ofinorganic materials, including metals or ceramics. Suitable metalsinclude chromium, rubidium, iron, zinc, selenium, nickel, gold, silver,platinum. Suitable ceramic materials in this embodiment include silicondioxide, titanium dioxide, aluminum oxide, ruthenium oxide and tinoxide. The core particles of this embodiment may be made from organicmaterials including carbon (diamond). Suitable polymers includepolystyrene, nylon and nitrocellulose. For this type of nanocrystallineparticle, particles made from tin oxide, titanium dioxide or carbon(diamond) are may also be used. A lipid micelle may be prepared by anymeans known in the art. For example micelles may be prepared accordingto the procedure of Baiselle and Millar (Biophys. Chem. 4: 355-361(1975)) or Corti et al. (Chem. Phys. Lipids 38: 197-214 (1981)) or Lopezet al. (FEBS Lett. 426: 314-318 (1998)) or Topchieva and Karezin (J.Colloid Interface Sci. 213: 29-35 (1999)) or Morein et al., (Nature 308:457-460 (1984)), which are all incorporated herein by reference.

The core particle may also be produced through a biological process,which may be natural or non-natural. By way of example, this type ofembodiment may includes a core particle comprising, or alternativelyconsisting of, a virus, virus-like particle, a bacterial pilus, a phage,a viral capsid particle or a recombinant form thereof In a more specificembodiment, the core particle may comprise, or alternatively consist of,recombinant proteins of Rotavirus, recombinant proteins of Norwalkvirus, recombinant proteins of Alphavirus, recombinant proteins whichform bacterial pili or pilus-like structures, recombinant proteins ofFoot and Mouth Disease virus, recombinant proteins of Retrovirus,recombinant proteins of Hepatitis B virus (e.g., a HBcAg), recombinantproteins of Tobacco mosaic virus, recombinant proteins of Flock HouseVirus, and recombinant proteins of human Papilomavirus.

Whether natural or non-natural, the core particle of the invention willgenerally have an organizer that is attached to the natural ornon-natural core particle by at least one covalent bond. The organizeris an element bound to a core particle in a non-random fashion thatprovides a nucleation site for creating an ordered and repetitiveantigen array. Ideally, but not necessarily, the organizer is associatedwith the core particle in a geometric order. Minimally, the organizercomprises a first attachment site.

In some embodiments of the invention, the ordered and repetitive arrayis formed by association between (1) either core particles ornon-natural molecular scaffolds and (2) an antigen or antigenicdeterminant. For example, bacterial pili or pilus-like structures areformed from proteins which are organized into ordered and repetitivestructures. Thus, in many instances, it will be possible to form orderedarrays of antigens or antigenic determinants by linking theseconstituents to bacterial pili or pili-like structures.

As previously stated, the organizer may be any element comprising atleast one first attachment site that is bound to a core particle by atleast one covalent bond. An organizer may be a protein, a polypeptide, apeptide, an amino acid (i.e., a residue of a protein, a polypeptide orpeptide), a sugar, a polynucleotide, a natural or synthetic polymer, asecondary metabolite or compound (biotin, fluorescein, retinol,digoxigenin, metal ions, phenylmethylsulfonylfluoride), or a combinationthereof, or a chemically reactive group thereof In a more specificembodiment, the organizer may comprise a first attachment sitecomprising an antigen, an antibody or antibody fragment, biotin, avidin,strepavidin, a receptor, a receptor ligand, a ligand, a ligand-bindingprotein, an interacting leucine zipper polypeptide, an amino group, achemical group reactive to an amino group; a carboxyl group, chemicalgroup reactive to a carboxyl group, a sulfhydryl group, a chemical groupreactive to a sulfhydryl group, or a combination thereof

In one embodiment, the core particle of the non-natural molecularscaffold comprises a virus, a bacterial pilus, a structure formed frombacterial pilin, a bacteriophage, a virus-like particle, a viral capsidparticle or a recombinant form thereof Any virus known in the art havingan ordered and repetitive coat and/or core protein structure may beselected as a non-natural molecular scaffold of the invention; examplesof suitable viruses include: sindbis and other alphaviruses; vesicularsomatitis virus; rhabdo-, (e.g. vesicular stomatitis virus), picorna-,toga-, orthomyxo-, polyoma-, parvovirus, rotavirus, Norwalk virus, footand mouth disease virus, a retrovirus, Hepatitis B virus, Tobacco mosaicvirus, flock house virus, human papilomavirus (for example, see Table 1in Bachman, M. F. and Zinkernagel, R. M., Immunol. Today 17: 553-558(1996)).

In one embodiment, the invention utilizes genetic engineering of a virusto create a fusion between an ordered and repetitive viral envelopeprotein and an organizer comprising a heterologous protein, peptide,antigenic determinant or a reactive amino acid residue of choice. Othergenetic manipulations known to those in the art may be included in theconstruction of the non-natural molecular scaffold; for example, it maybe desirable to restrict the replication ability of the recombinantvirus through genetic mutation. The viral protein selected for fusion tothe organizer (i.e., first attachment site) protein should have anorganized and repetitive structure. Such an organized and repetitivestructure include paracrystalline organizations with a spacing of 5-15nm on the surface of the virus. The creation of this type of fusionprotein will result in multiple, ordered and repetitive organizers onthe surface of the virus. Thus, the ordered and repetitive organizationof the first attachment sites resulting therefrom will reflect thenormal organization of the native viral protein.

As will be discussed in more detail herein, in another embodiment of theinvention, the non-natural molecular scaffold is a recombinantalphavirus, and more specifically, a recombinant Sinbis virus.Alphaviruses are positive stranded RNA viruses that replicate theirgenomic RNA entirely in the cytoplasm of the infected cell and without aDNA intermediate (Strauss, J. and Strauss, E., Microbiol Rev. 58:491-562 (1994)). Several members of the alphavirus family, Sindbis(Xiong, C. et al., Science 243: 1188-1191 (1989); Schlesinger, S.,Trends Biotechnol. 11: 18-22 (1993)), Semliki Forest Virus (SFV)(Liljeström, P. & Garoff, H., Bio/Technology 9: 1356-1361 (1991)) andothers (Davis, N. L. et al., Virology 171: 189-204 (1989)), havereceived considerable attention for use as virus-based expressionvectors for a variety of different proteins (Lundstrom, K., Curr. Opin.Biotechnol. 8: 578-582 (1997); Liljeström, P., Curr. Opin. Biotechnol.5: 495-500 (1994)) and as candidates for vaccine development. Recently,a number of patents have issued directed to the use of alphaviruses forthe expression of heterologous proteins and the development of vaccines(see U.S. Pat. Nos. 5,766,602; 5,792,462; 5,739,026; 5,789,245 and5,814,482). The construction of the alphaviral scaffold of the inventionmay be done by means generally known in the art of recombinant DNAtechnology, as described by the aforementioned articles, which areincorporated herein by reference.

A variety of different recombinant host cells can be utilized to producea viral-based core particle for antigen or antigenic determinantattachment. For example, Alphaviruses are known to have a wide hostrange; Sindbis virus infects cultured mammalian, reptilian, andamphibian cells, as well as some insect cells (Clark, H., J. Natl.Cancer Inst. 51: 645 (1973); Leake, C., J. Gen. Virol. 35: 335 (1977);Stollar, V. in THE TOGA VIRUSES, R. W. Schlesinger, Ed., Academic Press,(1980), pp. 583-621). Thus, numerous recombinant host cells can be usedin the practice of the invention. BHK, COS, Vero, HeLa and CHO cells areparticularly suitable for the production of heterologous proteinsbecause they have the potential to glycosylate heterologous proteins ina manner similar to human cells (Watson, E. et al., Glycobiology 4: 227,(1994)) and can be selected (Zang, M. et al., Bio/Technology 13: 389(1995)) or genetically engineered (Renner W. et al., Biotech. Bioeng. 4:476 (1995); Lee K. et al. Biotech. Bioeng. 50: 336 (1996)) to grow inserum-free medium, as well as in suspension.

Introduction of the polynucleotide vectors into host cells can beeffected by methods described in standard laboratory manuals (see, e.g.,Sambrook, J. et al., eds., MOLECULAR CLONING, A L ABORATORY MANUAL, 2nd.edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989), Chapter 9; Ausubel, F. et al., eds., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John H. Wiley & Sons, Inc. (1997), Chapter 16),including methods such as electroporation, DEAE-dextran mediatedtransfection, transfection. microinjection, cationic lipid-mediatedtransfection, transduction, scrape loading. ballistic introduction, andinfection. Methods for the introduction of exogenous DNA sequences intohost cells are discussed in Felgner, P. et al., U.S. Pat. No. 5,580,859.

Packaged RNA sequences can also be used to infect host cells. Thesepackaged RNA sequences can be introduced to host cells by adding them tothe culture medium. For example, the preparation of non-infectivealpahviral particles is described in a number of sources, including“Sindbis Expression System”, Version C (Invitrogen Catalog No. K750-1).

When mammalian cells are used as recombinant host cells for theproduction of viral-based core particles, these cells will generally begrown in tissue culture. Methods for growing cells in culture are wellknown in the art (see. e.g., Celis, J., ed., CELL BIOLOGY, AcademicPress, 2^(nd) edition, (1998); Sambrook, J. et al., eds., MOLECULARCLONING, A LABORATORY MANUAL, 2nd. edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel. F. et al.,eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John H. Wiley & Sons, Inc.(1997); Freshney, R., CULTURE OF ANIMAL CELLS, Alan R. Liss, Inc.(1983)).

As will be understood by those in the art, the first attachment site maybe or be a part of any suitable protein, polypeptide, sugar,polynucleotide, peptide (amino acid), natural or synthetic polymer, asecondary metabolite or combination thereof that may serve tospecifically attach the antigen or antigenic determinant of choice tothe non-natural molecular scaffold. In one embodiment, the attachmentsite is a protein or peptide that may be selected from those known inthe art. For example, the first attachment site may selected from thefollowing group: a ligand, a receptor, a lectin, avidin, streptavidin,biotin, an epitope such as an HA or T7 tag, Myc, Max, immunoglobulindomains and any other amino acid sequence known in the art that would beuseful as a first attachment site.

It should be further understood by those in the art that with anotherembodiment of the invention, the first attachment site may be createdsecondarily to the organizer (i.e., protein or polypeptide) utilized inconstructing the in-frame fusion to the capsid protein. For example, aprotein may be utilized for fusion to the envelope protein with an aminoacid sequence known to be glycosylated in a specific fashion, and thesugar moiety added as a result may then serve at the first attachmentsite of the viral scaffold by way of binding to a lectin serving as thesecondary attachment site of an antigen. Alternatively, the organizersequence may be biotinylated in vivo and the biotin moiety may serve asthe first attachment site of the invention, or the organizer sequencemay be subjected to chemical modification of distinct amino acidresidues in vitro, the modification serving as the first attachmentsite.

One specific embodiment of the invention utilizes the Sinbis virus. TheSinbis virus RNA genome is packaged into a capsid protein that issurrounded by a lipid bilayer containing three proteins called E1, E2,and E3. These so-called envelope proteins are glycoproteins, and theglycosylated portions are located on the outside of the lipid bilayer,where complexes of these proteins form the “spikes” that can be seen inelectron micrographs to project outward from the surface of the virus.In another embodiment of the invention, the first attachment site isselected to be the JUN or FOS leucine zipper protein domain that isfused in frame to the E2 envelope protein. However, it will be clear toall individuals in the art that other envelope proteins may be utilizedin the fusion protein construct for locating the first attachment sitein the non-natural molecular scaffold of the invention.

In a specific embodiment of the invention, the first attachment site isselected to be the JUN-FOS leucine zipper protein domain that is fusedin frame to the Hepatitis B capsid (core) protein (HBcAg). However, itwill be clear to all individuals in the art that other viral capsidproteins may be utilized in the fusion protein construct for locatingthe first attachment site in the non-natural molecular scaffold of theinvention.

In another specific embodiment of the invention, the first attachmentsite is selected to be a lysine or cysteine residue that is fused inframe to the HBcAg. However, it will be clear to all individuals in theart that other viral capsid or virus-like particles may be utilized inthe fusion protein construct for locating the first attachment in thenon-natural molecular scaffold of the invention.

Example 1 is provided to demonstrate the construction of an in-framefusion protein between the Sinbis virus E2 envelope protein and the JUNleucine zipper protein domain using the pTE5′2J vector of Hahn et al.(Proc. Natl. Acad. Sci. USA 89: 2679-2683 (1992)). The JUN amino acidsequence utilized for the first attachment site is the following:CGGRIARLEEKVKTLKAQNSE LASTANMLREQVAQLKQKVMNHVGC (SEQ ID NO:59). In thisinstance, the anticipated second attachment site on the antigen would bethe FOS leucine zipper protein domain and the amino acid sequence wouldbe the following: CGGLTDTLQAETDQVEDEKSALQTEIANLLKEKEKLEFILAAHGGC (SEQ IDNO:60)

These sequences are derived from the transcription factors JUN and FOS,each flanked with a short sequence containing a cysteine residue on bothsides. These sequences are known to interact with each other. Theoriginal hypothetical structure proposed for the JUN-FOS dimer assumedthat the hydrophobic side chains of one monomer interdigitate with therespective side chains of the other monomer in a zipper-like manner(Landschulz et al., Science 240: 1759-1764 (1988)). However, thishypothesis proved to be wrong, and these proteins are known to form ana-helical coiled coil (O'Shea et al., Science 243: 538-542 (1989);O'Shea et al., Cell 68: 699-708 (1992); Cohen & Parry, Trends Biochem.Sci. 11: 245-248 (1986)). Thus, the term “leucine zipper” is frequentlyused to refer to these protein domains for more historical thanstructural reasons. Throughout this patent, the term “leucine zipper” isused to refer to the sequences depicted above or sequences essentiallysimilar to the ones depicted above. The terms JUN and FOS are used forthe respective leucine zipper domains rather than the entire JUN and FOSproteins.

In one embodiment, the invention provides for the production of a Sinbisvirus E2-JUN scaffold using the pCYTts expression system (WO 99/50432).The pCYTts expression system provides novel expression vectors whichpermit tight regulation of gene expression in eucaryotic cells. The DNAvectors of this system are transcribed to form RNA molecules which arethen replicated by a temperature-sensitive replicase to form additionalRNA molecules. The RNA molecules produced by replication contain anucleotide sequence which may be translated to produce a protein ofinterest or which encode one or more untranslated RNA molecules. Thusthe expression system enables the production of recombinant Sinbis virusparticles.

Example 2 provides details on the production of the E2-JUN Sinbisnon-natural molecular scaffold of the invention. Additionally providedin Example 3 is another method for the production of recombinant E2-JUNSinbis virus scaffold using the pTE5′2JE2: JUN vector produced inExample 1. Thus the invention provides two means, the pCYTts expressionsystem (Example 2) and the pTE5′2J vector system (Example 3) by whichrecombinant Sinbis virus E2-JUN non-natural molecular scaffold may beproduced. An analysis of viral particles produced in each system isprovided in FIG. 1 and FIG. 2.

As previously stated, the invention includes viral-based core particleswhich comprise, or alternatively consist of, a virus, virus-likeparticle, a phage, a viral capsid particle or a recombinant form thereofSkilled artisans have the knowledge to produce such core particles andattach organizers thereto. By way of providing other examples, theinvention provides herein for the production of Hepatitis B virus-likeparticles and measles viral capsid particles as core particles (Examples17 to 22). In such an embodiment, the JUN leucine zipper protein domainor FOS leucine zipper protein domain may be used as an organizer, andhence as a first attachment site, for the non-natural molecular scaffoldof the invention.

Examples 23-29 provide details of the production of Hepatitis B coreparticles carrying an in-frame fused peptide with a reactive lysineresidue and antigens carrying a genetically fused cysteine residue, asfirst and second attachment site, respectively.

In other embodiments, the core particles used in compositions of theinvention are composed of a Hepatitis B capsid (core) protein (HBcAg),or fragment thereof, which has been modified to either eliminate orreduce the number of free cysteine residues. Zhou et al. (J. Virol. 66:5393-5398 (1992)) demonstrated that HBcAgs which have been modified toremove the naturally resident cysteine residues retain the ability toassociate and form multimeric structures. Thus, core particles suitablefor use in compositions of the invention include those comprisingmodified HBcAgs, or fragments thereof, in which one or more of thenaturally resident cysteine residues have been either deleted orsubstituted with another amino acid residue (e.g., a serine residue).

The HBcAg is a protein generated by the processing of a Hepatitis B coreantigen precursor protein. A number of isotypes of the HBcAg have beenidentified. For example, the HBcAg protein having the amino acidsequence shown in SEQ ID NO:132 is 183 amino acids in length and isgenerated by the processing of a 212 amino acid Hepatitis B core antigenprecursor protein. This processing results in the removal of 29 aminoacids from the N-terminus of the Hepatitis B core antigen precursorprotein. Similarly, the HBcAg protein having the amino acid sequenceshown in SEQ ID NO:134 is 185 amino acids in length and is generated bythe processing of a 214 amino acid Hepatitis B core antigen precursorprotein. The amino acid sequence shown in SEQ ID NO:134, as compared tothe amino acid sequence shown in SEQ ID NO:132, contains a two aminoacid insert at positions 152 and 153 in SEQ ID NO:134.

In most instances, vaccine compositions of the invention will beprepared using the processed form of a HBcAg (i.e., a HBcAg from whichthe N-terminal leader sequence (e.g., the first 29 amino acid residuesshown in SEQ ID NO:134) of the Hepatitis B core antigen precursorprotein have been removed).

Further, when HBcAgs are produced under conditions where processing willnot occur, the HBcAgs will generally be expressed in “processed” form.For example, bacterial systems, such as E. coli, generally do not removethe leader sequences of proteins which are normally expressed ineukaryotic cells. Thus, when an E. coli expression system is used toproduce HBcAgs of the invention, these proteins will generally beexpressed such that the N-terminal leader sequence of the Hepatitis Bcore antigen precursor protein is not present.

In one embodiment of the invention, a modified HBcAg comprising theamino acid sequence shown in SEQ ID NO:134, or subportion thereof, isused to prepare non-natural molecular scaffolds. In particular, modifiedHBcAgs suitable for use in the practice of the invention includeproteins in which one or more of the cysteine residues at positionscorresponding to positions 48, 61, 107 and 185 of a protein having theamino acid sequence shown in SEQ ID NO:134 have been either deleted orsubstituted with other amino acid residues (e.g., a serine residue). Asone skilled in the art would recognize, cysteine residues at similarlocations in HBcAg variants having amino acids sequences which differfrom that shown in SEQ ID NO:134 could also be deleted or substitutedwith other amino acid residues. The modified HBcAg variants can then beused to prepare vaccine compositions of the invention.

The present invention also includes HBcAg variants which have beenmodified to delete or substitute one or more additional cysteineresidues which are not found in polypeptides having the amino acidsequence shown in SEQ ID NO:134. Examples of such HBcAg variants havethe amino acid sequences shown in SEQ ID NOs: 90 and 132. These variantcontain cysteines residues at a position corresponding to amino acidresidue 147 in SEQ ID NO:134. Thus, the vaccine compositions of theinvention include compositions comprising HBcAgs in which cysteineresidues not present in the amino acid sequence shown in SEQ ID NO:134have been deleted.

Under certain circumstances (e.g., when a heterobifunctionalcross-linking reagent is used to attach antigens or antigenicdeterminants to the non-natural molecular scaffold), the presence offree cysteine residues in the HBcAg is believed to lead to covalentcoupling of toxic components to core particles, as well as thecross-linking of monomers to form undefined species.

Further, in many instances, these toxic components may not be detectablewith assays performed on compositions of the invention. This is sobecause covalent coupling of toxic components to the non-naturalmolecular scaffold would result in the formation of a population ofdiverse species in which toxic components are linked to differentcysteine residues, or in some cases no cysteine residues, of the HBcAgs.In other words, each free cysteine residue of each HBcAg will not becovalently linked to toxic components. Further, in many instances, noneof the cysteine residues of particular HBcAgs will be linked to toxiccomponents. Thus, the presence of these toxic components may bedifficult to detect because they would be present in a mixed populationof molecules. The administration to an individual of HBcAg speciescontaining toxic components, however, could lead to a potentiallyserious adverse reaction.

It is well known in the art that free cysteine residues can be involvedin a number of chemical side reactions. These side reactions includedisulfide exchanges, reaction with chemical substances or metabolitesthat are, for example, injected or formed in a combination therapy withother substances, or direct oxidation and reaction with nucleotides uponexposure to UV light. Toxic adducts could thus be generated, especiallyconsidering the fact that HBcAgs have a strong tendency to bind nucleicacids. Detection of such toxic products in antigen-capsid conjugateswould be difficult using capsids prepared using HBcAgs containing freecysteines and heterobifunctional cross-linkers, since a distribution ofproducts with a broad range of molecular weight would be generated. Thetoxic adducts would thus be distributed between a multiplicity ofspecies, which individually may each be present at low concentration,but reach toxic levels when together.

In view of the above, one advantage to the use of HBcAgs in vaccinecompositions which have been modified to remove naturally residentcysteine residues is that sites to which toxic species can bind whenantigens or antigenic determinants are attached to the non-naturalmolecular scaffold would be reduced in number or eliminated altogether.Further, a high concentration of cross-linker can be used to producehighly decorated particles without the drawback of generating aplurality of undefined cross-linked species of HBcAg monomers (i.e., adiverse mixture of cross-linked monomeric HbcAgs).

A number of naturally occurring HBcAg variants suitable for use in thepractice of the present invention have been identified. Yuan et al., (J.Virol. 73: 10122-10128 (1999)), for example, describe variants in whichthe isoleucine residue at position corresponding to position 97 in SEQID NO:134 is replaced with either a leucine residue or a phenylalanineresidue. The amino acid sequences of a number of HBcAg variants, as wellas several Hepatitis B core antigen precursor variants, are disclosed inGenBank reports AAF121240 (SEQ ID NO:89), AF121239 (SEQ ID NO:90),X85297 (SEQ ID NO:91), X02496 (SEQ ID NO:92), X85305 (SEQ ID NO:93),X85303 (SEQ ID NO:94), AF151735 (SEQ ID NO:95), X85259 (SEQ ID NO:96),X85286 (SEQ ID NO:97), X85260 (SEQ ID NO:98), X85317 (SEQ ID NO:99),X85298 (SEQ ID NO:100), AF043593 (SEQ ID NO:101), M20706 (SEQ IDNO:102), X85295 (SEQ ID NO:103), X80925 (SEQ ID NO:104), X85284 (SEQ IDNO:105), X85275 (SEQ ID NO:106), X72702 (SEQ ID NO:107), X85291 (SEQ IDNO:108), X65258 (SEQ ID NO:109), X85302 (SEQ ID NO:110), M32138 (SEQ IDNO:111), X85293 (SEQ ID NO:112), X85315 (SEQ ID NO:113), U95551 (SEQ IDNO:114), X85256 (SEQ ID NO:115), X85316 (SEQ ID NO:116), X85296 (SEQ IDNO:117), AB033559 (SEQ ID NO:118), X59795 (SEQ ID NO:119), X85299 (SEQID NO:120), X85307 (SEQ ID NO:121), X65257 (SEQ ID NO:122), X85311 (SEQID NO:123), X85301 (SEQ ID NO:124), X85314 (SEQ ID NO:125), X85287 (SEQID NO:126), X85272 (SEQ ID NO:127), X85319 (SEQ ID NO:128), AB010289(SEQ ID NO:129), X85285 (SEQ ID NO:130), AB010289 (SEQ ID NO:131),AF121242 (SEQ ID NO:132), M90520 (SEQ ID NO:135), P03153(SEQ ID NO:136),AF110999(SEQ ID NO:137), and M95589 (SEQ ID NO:138), the disclosures ofeach of which are incorporated herein by reference. These HBcAg variantsdiffer in amino acid sequence at a number of positions, including aminoacid residues which corresponds to the amino acid residues located atpositions 12, 13, 21, 22, 24, 29, 32, 33, 35, 38, 40, 42, 44, 45, 49,51, 57, 58, 59, 64, 66, 67, 69, 74, 77, 80, 81, 87, 92, 93, 97, 98, 100,103, 105, 106, 109, 113, 116, 121, 126, 130, 133, 135, 141, 147, 149,157, 176, 178, 182 and 183 in SEQ ID NO:134. The invention is alsodirected to amino acid sequences that are at least 65, 0, 75, 80, 85, 90or 95% identical to the above Hepatitis B viral capsid proteinsequences. HBcAgs suitable for use in the present invention may bederived from any organism so long as they are able to associate to forman ordered and repetitive antigen array.

As noted above, generally processed HBcAgs (i.e., those which lackleader sequences) will be used in the vaccine compositions of theinvention. Thus, when HBcAgs having amino acid sequence shown in SEQ IDNOs:136, 137, or 138 are used to prepare vaccine compositions of theinvention, generally 30, 35-43, or 35-43 amino acid residues at theN-terminus, respectively, of each of these proteins will be omitted.

The present invention includes vaccine compositions, as well as methodsfor using these compositions, which employ the above described variantHBcAgs for the preparation of non-natural molecular scaffolds.

Further included within the scope of the invention are additional HBcAgvariants which are capable of associating to form dimeric or multimericstructures. Thus, the invention further includes vaccine compositionscomprising HBcAg polypeptides comprising, or alternatively consistingof, amino acid sequences which are at least 80%, 85%, 90%, 95%, 97%, or99% identical to any of the amino acid sequences shown in SEQ IDNOs:89-132 and 134-138, and forms of these proteins which have beenprocessed, where appropriate, to remove the N-terminal leader sequence.

Whether the amino acid sequence of a polypeptide has an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, or 99% identical toone of the amino acid sequences shown in SEQ ID NOs:89-132 and 134-138,or a subportion thereof, can be determined conventionally using knowncomputer programs such the Bestfit program. When using Bestfit or anyother sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference amino acidsequence according to the present invention, the parameters are set suchthat the percentage of identity is calculated over the full length ofthe reference amino acid sequence and that gaps in homology of up to 5%of the total number of amino acid residues in the reference sequence areallowed.

The HBcAg variants and precursors having the amino acid sequences setout in SEQ ID NOs:89-132 and 134-136 are relatively similar to eachother. Thus, reference to an amino acid residue of a HBcAg variantlocated at a position which corresponds to a particular position in SEQID NO:134, refers to the amino acid residue which is present at thatposition in the amino acid sequence shown in SEQ ID NO:134. The homologybetween these HBcAg variants is for the most part high enough amongHepatitis B viruses that infect mammals so that one skilled in the artwould have little difficulty reviewing both the amino acid sequenceshown in SEQ ID NO:134 and that of a particular HBcAg variant andidentifying “corresponding” amino acid residues. For example, the HBcAgamino acid sequence shown in SEQ ID NO:135, which shows the amino acidsequence of a HBcAg derived from a virus which infect woodchucks, hasenough homology to the HBcAg having the amino acid sequence shown in SEQID NO:134 that it is readily apparent that a three amino acid residueinsert is present in SEQ ID NO:135 between amino acid residues 155 and156 of SEQ ID NO:134.

The HBcAgs of Hepatitis B viruses which infect snow geese and ducksdiffer enough from the amino acid sequences of HBcAgs of Hepatitis Bviruses which infect mammals that alignment of these forms of thisprotein with the amino acid sequence shown in SEQ ID NO:134 isdifficult. However, the invention includes vaccine compositions whichcomprise HBcAg variants of Hepatitis B viruses which infect birds, aswells as vaccine compositions which comprise fragments of these HBcAgvariants. HBcAg fragments suitable for use in preparing vaccinecompositions of the invention include compositions which containpolypeptide fragments comprising, or alternatively consisting of, aminoacid residues selected from the group consisting of 36-240, 36-269,44-240, 44-269, 36-305, and 44-305 of SEQ ID NO:137 or 36-240, 36-269,44-240, 44-269,36-305, and 44-305 of SEQ ID NO:138. As one skilled inthe art would recognize, one, two, three or more of the cysteineresidues naturally present in these polypeptides (e.g., the cysteineresidues at position 153 is SEQ ID NO:137 or positions 34, 43, and 196in SEQ ID NO:138) could be either substituted with another amino acidresidue or deleted prior to their inclusion in vaccine compositions ofthe invention.

In one embodiment, the cysteine residues at positions 48 and 107 of aprotein having the amino acid sequence shown in SEQ ID NO:134 aredeleted or substituted with another amino acid residue but the cysteineat position 61 is left in place. Further, the modified polypeptide isthen used to prepare vaccine compositions of the invention.

As set out below in Example 31, the cysteine residues at positions 48and 107, which are accessible to solvent, may be removed, for example,by site-directed mutagenesis. Further, the inventors have found that theCys-48-Ser, Cys-107-Ser HBcAg double mutant constructed as described inExample 31 can be expressed in E. coli.

As discussed above, the elimination of free cysteine residues reducesthe number of sites where toxic components can bind to the HBcAg, andalso eliminates sites where cross-linking of lysine and cysteineresidues of the same or of neighboring HBcAg molecules can occur. Thecysteine at position 61, which is involved in dimer formation and formsa disulfide bridge with the cysteine at position 61 of another HBcAg,will normally be left intact for stabilization of HBcAg dimers andmultimers of the invention.

As shown in Example 32, cross-linking experiments performed with (1)HBcAgs containing free cysteine residues and (2) HBcAgs whose freecysteine residues have been made unreactive with iodacetamide, indicatethat free cysteine residues of the HBcAg are responsible forcross-linking between HBcAgs through reactions betweenheterobifunctional cross-linker derivatized lysine side chains, and freecysteine residues. Example 32 also indicates that cross-linking of HBcAgsubunits leads to the formation of high molecular weight species ofundefined size which cannot be resolved by SDS-polyacrylamide gelelectrophoresis.

When an antigen or antigenic determinant is linked to the non-naturalmolecular scaffold through a lysine residue, it may be advantageous toeither substitute or delete one or both of the naturally resident lysineresidues located at positions corresponding to positions 7 and 96 in SEQID NO:134, as well as other lysine residues present in HBcAg variants.The elimination of these lysine residues results in the removal ofbinding sites for antigens or antigenic determinants which could disruptthe ordered array and should improve the quality and uniformity of thefinal vaccine composition.

In many instances, when both of the naturally resident lysine residuesat positions corresponding to positions 7 and 96 in SEQ ID NO:134 areeliminated, another lysine will be introduced into the HBcAg as anattachment site for an antigen or antigenic determinant. Methods forinserting such a lysine residue are set out, for example, in Example 23below. It will often be advantageous to introduce a lysine residue intothe HBcAg when, for example, both of the naturally resident lysineresidues at positions corresponding to positions 7 and 96 in SEQ IDNO:134 are altered and one seeks to attach the antigen or antigenicdeterminant to the non-natural molecular scaffold using aheterobifunctional cross-linking agent.

The C-terminus of the HBcAg has been shown to direct nuclearlocalization of this protein. (Eckhardt et al., J. Virol. 65: 575-582(1991).) Further, this region of the protein is also believed to conferupon the HBcAg the ability to bind nucleic acids.

In some embodiments, vaccine compositions of the invention will containHBcAgs which have nucleic acid binding activity (e.g., which contain anaturally resident HBcAg nucleic acid binding domain). HBcAgs containingone or more nucleic acid binding domains are useful for preparingvaccine compositions which exhibit enhanced T-cell stimulatory activity.Thus, the vaccine compositions of the invention include compositionswhich contain HBcAgs having nucleic acid binding activity. Furtherincluded are vaccine compositions, as well as the use of suchcompositions in vaccination protocols, where HBcAgs are bound to nucleicacids. These HBcAgs may bind to the nucleic acids prior toadministration to an individual or may bind the nucleic acids afteradministration.

In other embodiments, vaccine compositions of the invention will containHBcAgs from which the C-terminal region (e.g., amino acid residues145-185 or 150-185 of SEQ ID NO:134) has been removed and do not bindnucleic acids. Thus, additional modified HBcAgs suitable for use in thepractice of the present invention include C-terminal truncation mutants.Suitable truncation mutants include HBcAgs where 1, 5, 10, 15, 20, 25,30, 34, 35, 36, 37, 38, 39 40, 41, 42 or 48 amino acids have beenremoved from the C-terminus.

HBcAgs suitable for use in the practice of the present invention alsoinclude N-terminal truncation mutants. Suitable truncation mutantsinclude modified HBcAgs where 1, 2, 5, 7, 9, 10, 12, 14, 15, and 17amino acids have been removed from the N-terminus.

Further HBcAgs suitable for use in the practice of the present inventioninclude N- and C-terminal truncation mutants. Suitable truncationmutants include HBcAgs where 1, 2, 5, 7, 9, 10, 12, 14, 15, and 17 aminoacids have been removed from the N-terminus and 1, 5, 10, 15, 20, 25,30, 34, 35, 36, 37, 38, 39 40, 41, 42 or 48 amino acids have beenremoved from the C-terminus.

The invention further includes vaccine compositions comprising HBcAgpolypeptides comprising, or alternatively consisting of, amino acidsequences which are at least 80%, 85%, 90%, 95%, 97%, or 99% identicalto the above described truncation mutants.

As discussed above, in certain embodiments of the invention, a lysineresidue is introduced as a first attachment site into a polypeptidewhich forms the non-natural molecular scaffold. In preferredembodiments, vaccine compositions of the invention are prepared using aHBcAg comprising, or alternatively consisting of, amino acids 1-144 oramino acids 1-149 of SEQ ID NO:134 which is modified so that the aminoacids corresponding to positions 79 and 80 are replaced with a peptidehaving the amino acid sequence of Gly-Gly-Lys-Gly-Gly (SEQ ID NO:158)and the cysteine residues at positions 48 and 107 are either deleted orsubstituted with another amino acid residue, while the cysteine atposition 61 is left in place. The invention further includes vaccinecompositions comprising corresponding fragments of polypeptides havingamino acid sequences shown in any of SEQ ID NOs:89-132 and 135-136 whichalso have the above noted amino acid alterations.

The invention further includes vaccine compositions comprising fragmentsof a HBcAg comprising, or alternatively consisting of, an amino acidsequence other than that shown in SEQ ID NO:134 from which a cysteineresidue not present at corresponding location in SEQ ID NO:134 has beendeleted. One example of such a fragment would be a polypeptidecomprising, or alternatively consisting of, amino acids amino acids1-149 of SEQ ID NO:132 where the cysteine residue at position 147 hasbeen either substituted with another amino acid residue or deleted.

The invention further includes vaccine compositions comprising HBcAgpolypeptides comprising, or alternatively consisting of, amino acidsequences which are at least 80%, 85%, 90%, 95%, 97%, or 99% identicalto amino acids 1-144 or 1-149 of SEQ ID NO:134 and correspondingsubportions of a polypeptide comprising an amino acid sequence shown inany of SEQ ID NOs:89-132 or 134-136, as well as to amino acids 1-147 or1-152 of SEQ ID NO:158.

The invention also includes vaccine compositions comprising HBcAgpolypeptides comprising, or alternatively consisting of, amino acidsequences which are at least 80%, 85%, 90%, 95%, 97%, or 99% identicalto amino acids 36-240, 36-269, 44-240, 44-269, 36-305, and 44-305 of SEQID NO:137 or 36-240, 36-269, 44-240, 44-269, 36-305, and 44-305 of SEQID NO:138.

Vaccine compositions of the invention may comprise mixtures of differentHBcAgs. Thus, these vaccine compositions may be composed of HBcAgs whichdiffer in amino acid sequence. For example, vaccine compositions couldbe prepared comprising a “wild-type” HBcAg and a modified HBcAg in whichone or more amino acid residues have been altered (e.g., deleted,inserted or substituted). In most applications, however, only one typeof a HBcAg, or at least HBcAgs having essentially equivalent firstattachment sites, will be used because vaccines prepared using suchHBcAgs will present highly ordered and repetitive arrays of antigens orantigenic determinants. Further, preferred vaccine compositions of theinvention are those which present highly ordered and repetitive antigenarrays.

The invention further includes vaccine compositions where thenon-natural molecular scaffold is prepared using a HBcAg fused toanother protein. As discussed above, one example of such a fusionprotein is a HBcAg/FOS fusion. Other examples of HBcAg fusion proteinssuitable for use in vaccine compositions of the invention include fusionproteins where an amino acid sequence has been added which aids in theformation and/or stabilization of HBcAg dimers and multimers. Thisadditional amino acid sequence may be fused to either the N- orC-terminus of the HBcAg. One example, of such a fusion protein is afusion of a HBcAg with the GCN4 helix region of Saccharomyces cerevisiae(GenBank Accession No. P03069 (SEQ ID NO:154)).

The helix domain of the GCN4 protein forms homodimers via non-covalentinteractions which can be used to prepare and stabilize HBcAg dimers andmultimers.

In one embodiment, the invention provides vaccine compositions preparedusing HBcAg fusions proteins comprising a HBcAg, or fragment thereof,with a GCN4 polypeptide having the sequence of amino acid residues 227to 276 in SEQ ID NO:154 fused to the C-terminus. This GCN4 polypeptidemay also be fused to the N-terminus of the HbcAg.

HBcAg/src homology 3 (SH3) domain fusion proteins could also be used toprepare vaccine compositions of the invention. SH3 domains arerelatively small domains found in a number of proteins which confer theability to interact with specific proline-rich sequences in proteinbinding partners (see McPherson, Cell Signal 11: 229-238 (1999).HBcAg/SH3 fusion proteins could be used in several ways. First, the SH3domain could form a first attachment site which interacts with a secondattachment site of the antigen or antigenic determinant. Similarly, aproline rich amino acid sequence could be added to the HBcAg and used asa first attachment site for an SH3 domain second attachment site of anantigen or antigenic determinant. Second, the SH3 domain could associatewith proline rich regions introduced into HBcAgs. Thus, SH3 domains andproline rich SH3 interaction sites could be inserted into either thesame or different HBcAgs and used to form and stabilized dimers andmultimers of the invention.

In other embodiments, a bacterial pilin, a subportion of a bacterialpilin, or a fusion protein which contains either a bacterial pilin orsubportion thereof is used to prepare vaccine compositions of theinvention. Examples of pilin proteins include pilins produced byEscherichia coli, Haemophilus influenzae, Neisseria meningitidis,Neisseria gonorrhoeae, Caulobacter crescentus, Pseudomonas stutzeri, andPseudomonas aeruginosa. The amino acid sequences of pilin proteinssuitable for use with the present invention include those set out inGenBank reports AJ000636 (SEQ ID NO:139), AJ132364 (SEQ ID NO:140),AF229646 (SEQ ID NO:141), AF051814 (SEQ ID NO:142), AF051815 (SEQ IDNO:143), and X00981 (SEQ ID NO:155), the entire disclosures of which areincorporated herein by reference.

Bacterial pilin proteins are generally processed to remove N-terminalleader sequences prior to export of the proteins into the bacterialperiplasm. Further, as one skilled in the art would recognize, bacterialpilin proteins used to prepare vaccine compositions of the inventionwill generally not have the naturally present leader sequence.

One specific example of a pilin protein suitable for use in the presentinvention is the P-pilin of E. coli (GenBank report AF237482 (SEQ IDNO:144)).

An example of a Type-1 E. coli pilin suitable for use with the inventionis a pilin having the amino acid sequence set out in GenBank reportP04128 (SEQ ID NO:146), which is encoded by nucleic acid having thenucleotide sequence set out in GenBank report M27603 (SEQ ID NO:145).The entire disclosures of these GenBank reports are incorporated hereinby reference. Again, the mature form of the above referenced proteinwould generally be used to prepare vaccine compositions of theinvention. Another example of a pilin protein is SEQ ID NO: 184, whichis identical to SEQ ID NO:146, except that in SEQ ID NO:146, amino acid20 is threonine, but in SEQ ID NO:184, amino acid 20 is alanine.

Bacterial pilins or pilin subportions suitable for use in the practiceof the present invention will generally be able to associate to formnon-natural molecular scaffolds.

Methods for preparing pili and pilus-like structures in vitro are knownin the art. Bullitt et al, Proc. Natl. Acad. Sci. USA 93: 12890-12895(1996), for example, describe the in vitro reconstitution of E. coliP-pili subunits. Further, Eshdat et al., J. Bacteriol. 148: 308-314(1981) describe methods suitable for dissociating Type-1 pili of E. coliand the reconstitution of pili. In brief, these methods are as follows:pili are dissociated by incubation at 37° C. in saturated guanidinehydrochloride. Pilin proteins are then purified by chromatography, afterwhich pilin dimers are formed by dialysis against 5 mMtris(hydroxymethyl) aminomethane hydrochloride (pH 8.0). Eshdat et al.also found that pilin dimers reassemble to form pili upon dialysisagainst the 5 mM tris(hydroxymethyl) aminomethane (pH 8.0) containing 5mM MgCl₂.

Further, using, for example, conventional genetic engineering andprotein modification methods, pilin proteins may be modified to containa first attachment site to which an antigen or antigenic determinant islinked through a second attachment site. Alternatively, antigens orantigenic determinants can be directly linked through a secondattachment site to amino acid residues which are naturally resident inthese proteins. These modified pilin proteins may then be used invaccine compositions of the invention.

Bacterial pilin proteins used to prepare vaccine compositions of theinvention may be modified in a manner similar to that described hereinfor HBcAg. For example, cysteine and lysine residues may be eitherdeleted or substituted with other amino acid residues and firstattachment sites may be added to these proteins. Further, pilin proteinsmay either be expressed in modified form or may be chemically modifiedafter expression. Similarly, intact pili may be harvested from bacteriaand then modified chemically.

In another embodiment, pili or pilus-like structures are harvested frombacteria (e.g., E. coli) and used to form vaccine compositions of theinvention. One example of pili suitable for preparing vaccinecompositions is the Type-1 pilus of E. coli, which is formed from pilinmonomers having the amino acid sequence set out in SEQ ID NO:146.

A number of methods for harvesting bacterial pili are known in the art.Bullitt and Makowski (Biophys. J. 74: 623-632 (1998)), for example,describe a pilus purification method for harvesting P-pili from E. coli.According to this method, pili are sheared from hyperpiliated E. colicontaining a P-pilus plasmid and purified by cycles of solubilizationand MgCl₂ (1.0 M) precipitation. A similar purification method is setout below in Example 33.

Once harvested, pili or pilus-like structures may be modified in avariety of ways. For example, a first attachment site can be added tothe pili to which antigens or antigen determinants may be attachedthrough a second attachment site. In other words, bacterial pili orpilus-like structures can be harvested and modified to form non-naturalmolecular scaffolds.

Pili or pilus-like structures may also be modified by the attachment ofantigens or antigenic determinants in the absence of a non-naturalorganizer. For example, antigens or antigenic determinants could belinked to naturally occurring cysteine resides or lysine residues. Insuch instances, the high order and repetitiveness of a naturallyoccurring amino acid residue would guide the coupling of the antigens orantigenic determinants to the pili or pilus-like structures. Forexample, the pili or pilus-like structures could be linked to the secondattachment sites of the antigens or antigenic determinants using aheterobifunctional cross-linking agent.

When structures which are naturally synthesized by organisms (e.g.,pili) are used to prepare vaccine compositions of the invention, it willoften be advantageous to genetically engineer these organisms so thatthey produce structures having desirable characteristics. For example,when Type-1 pili of E. coli are used, the E. coli from which these piliare harvested may be modified so as to produce structures with specificcharacteristics. Examples of possible modifications of pilin proteinsinclude the insertion of one or more lysine residues, the deletion orsubstitution of one or more of the naturally resident lysine residues,and the deletion or substitution of one or more naturally residentcysteine residues (e.g., the cysteine residues at positions 44 and 84 inSEQ ID NO:146).

Further, additional modifications can be made to pilin genes whichresult in the expression products containing a first attachment siteother than a lysine residue (e.g., a FOS or JUN domain). Of course,suitable first attachment sites will generally be limited to those whichdo not prevent pilin proteins from forming pili or pilus-like structuressuitable for use in vaccine compositions of the invention.

Pilin genes which naturally reside in bacterial cells can be modified invivo (e.g., by homologous recombination) or pilin genes with particularcharacteristics can be inserted into these cells. For examples, pilingenes could be introduced into bacterial cells as a component of eithera replicable cloning vector or a vector which inserts into the bacterialchromosome. The inserted pilin genes may also be linked to expressionregulatory control sequences (e.g., a lac operator).

In most instances, the pili or pilus-like structures used in vaccinecompositions of the invention will be composed of single type of a pilinsubunit. Pili or pilus-like structures composed of identical subunitswill generally be used because they are expected to form structureswhich present highly ordered and repetitive antigen arrays.

However, the compositions of the invention also include vaccinescomprising pili or pilus-like structures formed from heterogenous pilinsubunits. The pilin subunits which form these pili or pilus-likestructures can be expressed from genes naturally resident in thebacterial cell or may be introduced into the cells. When a naturallyresident pilin gene and an introduced gene are both expressed in a cellwhich forms pili or pilus-like structures, the result will generally bestructures formed from a mixture of these pilin proteins. Further, whentwo or more pilin genes are expressed in a bacterial cell, the relativeexpression of each pilin gene will typically be the factor whichdetermines the ratio of the different pilin subunits in the pili orpilus-like structures.

When pili or pilus-like structures having a particular composition ofmixed pilin subunits is desired, the expression of at least one of thepilin genes can be regulated by a heterologous, inducible promoter. Suchpromoters, as well as other genetic elements, can be used to regulatethe relative amounts of different pilin subunits produced in thebacterial cell and, hence, the composition of the pili or pilus-likestructures.

In additional, while in most instances the antigen or antigenicdeterminant will be linked to bacterial pili or pilus-like structures bya bond which is not a peptide bond, bacterial cells which produce pilior pilus-like structures used in the compositions of the invention canbe genetically engineered to generate pilin proteins which are fused toan antigen or antigenic determinant. Such fusion proteins which formpili or pilus-like structures are suitable for use in vaccinecompositions of the invention.

The inventors surprisingly found that bacterial Pili induced an antibodyresponse dominated by the IgG1 isotype in mince. This type of antibodiesis indicative for a Th2 response. Moreover, antigens coupled to Pilialso induced a IgG1 response indicating that coupling of antigens toPili was sufficient for induction of antigen-specific Th2 responses.

B. Construction of an Antigen or Antigenic Determinant with a SecondAttachment Site

The second element in the compositions of the invention is an antigen orantigenic determinant possessing at least one second attachment sitecapable of association through at least one non-peptide bond to thefirst attachment site of the non-natural molecular scaffold. Theinvention provides for compositions that vary according to the antigenor antigenic determinant selected in consideration of the desiredtherapeutic effect. Other compositions are provided by varying themolecule selected for the second attachment site.

However, when bacterial pili, or pilus-like structures, pilin proteinsare used to prepare vaccine compositions of the invention, antigens orantigenic determinants may be attached to pilin proteins by theexpression of pilin/antigen fusion proteins. Antigen and antigenicdeterminants may also be attached to bacterial pili, or pilus-likestructures, pilin proteins through non-peptide bonds.

Antigens of the invention may be selected from the group consisting ofthe following: (a) proteins suited to induce an immune response againstcancer cells; (b) proteins suited to induce an immune response againstinfectious diseases; (c) proteins suited to induce an immune responseagainst allergens, (d) proteins suited to induce an immune response infarm animals, and (e) fragments (e.g., a domain) of any of the proteinsset out in (a)-(d).

In one specific embodiment of the invention, the antigen or antigenicdeterminant is one that is useful for the prevention of infectiousdisease. Such treatment will be useful to treat a wide variety ofinfectious diseases affecting a wide range of hosts, e.g., human, cow,sheep, pig, dog, cat, other mammalian species and non-mammalian speciesas well. Treatable infectious diseases are well known to those skilledin the art, examples include infections of viral etiology such as HIV,influenza, Herpes, viral hepatitis, Epstein Bar, polio, viralencephalitis, measles, chicken pox, etc.; or infections of bacterialetiology such as pneumonia, tuberculosis, syphilis, etc.; or infectionsof parasitic etiology such as malaria, trypanosomiasis, leishmaniasis,trichomoniasis, amoebiasis, etc. Thus, antigens or antigenicdeterminants selected for the compositions of the invention will be wellknown to those in the medical art; examples of antigens or antigenicdeterminants include the following: the HIV antigens gp140 and gp160;the influenaza antigens hemagglutinin and neuraminidase, Hepatitis Bsurface antigen, circumsporozoite protein of malaria.

In another specific embodiment, compositions of the invention are animmunotherapeutic that may be used for the treatment of allergies orcancer.

The selection of antigens or antigenic determinants for compositions andmethods of treatment for allergies would be known to those skilled inthe medical art treating such disorders; representative examples of thistype of antigen or antigenic determinant include the following: beevenom phospholipase A₂, Bet v I(birch pollen allergen), 5 Dol m V(white-faced hornet venom allergen), Der p I(House dust mite allergen).

The selection of antigens or antigenic determinants for compositions andmethods of treatment for cancer would be known to those skilled in themedical art treating such disorders; representative examples of thistype of antigen or antigenic determinant include the following: Her2(breast cancer), GD2 (neuroblastoma), EGF-R (malignant glioblastoma),CEA (medullary thyroid cancer), CD52 (leukemia).

In a particular embodiment of the invention, the antigen or antigenicdeterminant is selected from the group consisting of (a) a recombinantprotein of HIV, (b) a recombinant protein of Influenza virus, (c) arecombinant protein of Hepatitis B virus, (d) a recombinant protein ofToxoplasma, (e) a recombinant protein of Plasmodiun falciparum, (f) arecombinant protein of Plasmodium vivax, (g) a recombinant protein ofPlasmodium ovale, (h) a recombinant protein of Plasmodium malariae, (i)a recombinant protein of breast cancer cells, (j) a recombinant proteinof kidney cancer cells, (k) a recombinant protein of prostate cancercells, (l) a recombinant protein of skin cancer cells, (m) a recombinantprotein of brain cancer cells, (n) a recombinant protein of leukemiacells, (o) a recombinant profiling, (p) a recombinant protein of beesting allergy, (q) a recombinant proteins of nut allergy, (r) arecombinant proteins of food allergies, (s) recombinant proteins ofasthma, (t) a recombinant protein of Chlamydia, and (u) a fragment ofany of the proteins set out in (a)-(t).

Once the antigen or antigenic determinant of the composition is chosen,at least one second attachment site may be added to the molecule inpreparing to construct the organized and repetitive array associatedwith the non-natural molecular scaffold of the invention. Knowledge ofwhat will constitute an appropriate second attachment site will be knownto those skilled in the art. Representative examples of secondattachment sites include, but are not limited to, the following: anantigen, an antibody or antibody fragment, biotin, avidin, strepavidin,a receptor, a receptor ligand, a ligand, a ligand-binding protein, aninteracting leucine zipper polypeptide, an amino group, a chemical groupreactive to an amino group; a carboxyl group, chemical group reactive toa carboxyl group, a sulfhydryl group, a chemical group reactive to asulfhydryl group, or a combination thereof

The association between the first and second attachment sites will bedetermined by the characteristics of the respective molecules selectedbut will comprise at least one non-peptide bond. Depending upon thecombination of first and second attachment sites, the nature of theassociation may be covalent, ionic, hydrophobic, polar, or a combinationthereof

In one embodiment of the invention, the second attachment site may bethe FOS leucine zipper protein domain or the JUN leucine zipper proteindomain.

In a more specific embodiment of the invention, the second attachmentsite selected is the FOS leucine zipper protein domain, which associatesspecifically with the JUN leucine zipper protein domain of thenon-natural molecular scaffold of the invention. The association of theJUN and FOS leucine zipper protein domains provides a basis for theformation of an organized and repetitive antigen or antigenicdeterminant array on the surface of the scaffold. The FOS leucine zipperprotein domain may be fused in frame to the antigen or antigenicdeterminant of choice at either the amino terminus, carboxyl terminus orinternally located in the protein if desired.

Several FOS fusion constructs are provided for exemplary purposes. Humangrowth hormone (Example 4), bee venom phospholipase A₂ (PLA) (Example9), ovalbumin (Example 10) and HIV gp140 (Example 12).

In order to simplify the generation of FOS fusion constructs, severalvectors are disclosed that provide options for antigen or antigenicdeterminant design and construction (see Example 6). The vectors pAV1-4were designed for the expression of FOS fusion in E. coli; the vectorspAV5 and pAV6 were designed for the expression of FOS fusion proteins ineukaryotic cells. Properties of these vectors are briefly described:

1. pAV1: This vector was designed for the secretion of fusion proteinswith FOS at the C-terminus into the E. coli periplasmic space. The geneof interest (g.o.i.) may be ligated into the Stul/NotI sites of thevector.

2. pAV2: This vector was designed for the secretion of fusion proteinswith FOS at the N-terminus into the E. coli periplasmic space. The geneof interest (g.o.i.) ligated into the NotI/EcoRV (or NotI/HindIII) sitesof the vector.

3. pAV3: This vector was designed for the cytoplasmic production offusion proteins with FOS at the C-terminus in E. coli. The gene ofinterest (g.o.i.) may be ligated into the EcoRV/NotI sites of thevector.

4. pAV4: This vector is designed for the cytoplasmic production offusion proteins with FOS at the N-terminus in E. coli. The gene ofinterest (g.o.i.) may be ligated into the NotI/EcoRV (or NotI/HindIII)sites of the vector. The N-terminal methionine residue isproteolytically removed upon protein synthesis (Hirel et al., Proc.Natl. Acad. Sci. USA 86: 8247-8251 (1989)).

5. pAV5: This vector was designed for the eukaryotic production offusion proteins with FOS at the C-terminus. The gene of interest(g.o.i.) may be inserted between the sequences coding for the hGH signalsequence and the FOS domain by ligation into the Eco47III/NotI sites ofthe vector. Alternatively, a gene containing its own signal sequence maybe fused to the FOS coding region by ligation into the StuL/NotI sites.

6. pAV6: This vector was designed for the eukaryotic production offusion proteins with FOS at the N-terminus. The gene of interest(g.o.i.) may be ligated into the NotI/StuI (or NotI/HindIII) sites ofthe vector.

As will be understood by those skilled in the art, the construction of aFOS-antigen or -antigenic determinant fusion protein may include theaddition of certain genetic elements to facilitate production of therecombinant protein. Example 4 provides guidance for the addition ofcertain E. coli regulatory elements for translation, and Example 7provides guidance for the addition of a eukaryotic signal sequence.Other genetic elements may be selected, depending on the specific needsof the practioner.

The invention is also seen to include the production of the FOS-antigenor FOS-antigenic determinant fusion protein either in bacterial (Example5) or eukaryotic cells (Example 8). The choice of which cell type inwhich to express the fusion protein is within the knowledge of theskilled artisan, depending on factors such as whether post-translationalmodifications are an important consideration in the design of thecomposition.

As noted previously, the invention discloses various methods for theconstruction of a FOS-antigen or FOS-antigenic determinant fusionprotein through the use of the pAV vectors. In addition to enablingprokaryotic and eukaryotic expression, these vectors allow thepractitioner to choose between N- and C-terminal addition to the antigenof the FOS leucine zipper protein domain. Specific examples are providedwherein N- and C-terminal FOS fusions are made to PLA (Example 9) andovalbumin (Example 10). Example 11 demonstrates the purification of thePLA and ovalbumin FOS fusion proteins.

In a more specific embodiment, the invention is drawn to an antigen orantigenic determinant encoded by the HIV genome. More specifically, theHIV antigen is gp140. As provided for in Examples 11-15, HIV gp140 maybe created with a FOS leucine zipper protein domain and the fusionprotein synthesized and purified for attachment to the non-naturalmolecular scaffold of the invention. As one skilled in the art wouldknow, other HIV antigens or antigenic determinants may be used in thecreation of a composition of the invention.

In a more specific embodiment of the invention, the second attachmentsite selected is a cysteine residue, which associates specifically witha lysine residue of the non-natural molecular scaffold of the invention.The chemical linkage of the lysine residue (Lys) and cysteine residue(Cys) provides a basis for the formation of an organized and repetitiveantigen or antigenic determinant array on the surface of the scaffold.The cysteine residue may be engineered in frame to the antigen orantigenic determinant of choice at either the amino terminus, carboxylterminus or internally located in the protein if desired. By way ofexample, PLA and HIV gp140 are provided with a cysteine residue forlinkage to a lysine residue first attachment site.

C. Preparation of the Alpha Vaccine Particles

The invention provides novel compositions and methods for theconstruction of ordered and repetitive antigen arrays. As one of skillin the art would know, the conditions for the assembly of the orderedand repetitive antigen array depend to a large extent on the specificchoice of the first attachment site of the non-natural molecularscaffold and the specific choice of the second attachment site of theantigen or antigenic determinant. Thus, practitioner choice in thedesign of the composition (i.e., selection of the first and secondattachment sites, antigen and non-natural molecular scaffold) willdetermine the specific conditions for the assembly of the Alpha Vaccineparticle (the ordered and repetitive antigen array and non-naturalmolecular scaffold combined). Information relating to assembly of theAlphaVaccine particle is well within the working knowledge of thepractitioner, and numerous references exist to aid the practitioner(e.g., Sambrook, J. et al., eds., MOLECULAR CLONING, A LABORATORYMANUAL, 2nd. edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989); Ausubel, F. et al., eds., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John H. Wiley & Sons, Inc. (1997); Celis, J., ed.,CELL BIOLGY, Academic Press, 2^(nd) edition, (1998); Harlow,. E. andLane, D., “Antibodies: A Laboratory Manual,” Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1988), all of which areincorporated herein by reference.

In a specific embodiment of the invention, the JUN and FOS leucinezipper protein domains are utilized for the first and second attachmentsites of the invention, respectively. In the preparation of AlphaVaccineparticles, antigen must be produced and purified under conditions topromote assembly of the ordered and repetitive antigen array onto thenon-natural molecular scaffold. In the particular JUN/FOS leucine zipperprotein domain embodiment, the FOS-antigen or FOS-antigenic determinantshould be treated with a reducing agent (e.g., Dithiothreitol (DTT)) toreduce or eliminate the incidence of disulfide bond formation (Example15).

For the preparation of the non-natural molecular scaffold (i.e.,recombinant Sinbis virus) of the JUN/FOS leucine zipper protein domainembodiment, recombinant E2-JUN viral particles should be concentrated,neutralized and treated with reducing agent (see Example 16).

Assembly of the ordered and repetitive antigen array in the JUN/FOSembodiment is done in the presence of a redox shuffle. E2-JUN viralparticles are combined with a 240 fold molar excess of FOS-antigen orFOS-antigenic determinant for 10 hours at 4° C. Subsequently, theAlphaVaccine particle is concentrated and purified by chromatography(Example 16).

In another embodiment of the invention, the coupling of the non-naturalmolecular scaffold to the antigen or antigenic determinant may beaccomplished by chemical cross-linking. In a specific embodiment, thechemical agent is a heterobifunctional cross-linking agent such asε-maleimidocaproic acid N-hydroxysuccinimide ester (Tanimori et al., J.Pharm. Dyn. 4: 812 (1981); Fujiwara et al., J. Immunol. Meth. 45: 195(1981)), which contains (1) a succinimide group reactive with aminogroups and (2) a maleimide group reactive with SH groups. A heterologousprotein or polypeptide of the first attachment site may be engineered tocontain one or more lysine residues that will serve as a reactive moietyfor the succinimide portion of the heterobifunctional cross-linkingagent. Once chemically coupled to the lysine residues of theheterologous protein, the maleimide group of the heterobifunctionalcross-linking agent will be available to react with the SH group of acysteine residue on the antigen or antigenic determinant. Antigen orantigenic determinant preparation in this instance may require theengineering of a cysteine residue into the protein or polypeptide chosenas the second attachment site so that it may be reacted to the freemaleimide function on the cross-linking agent bound to the non-naturalmolecular scaffold first attachment sites. Thus, in such an instance,the heterobifunctional cross-linking agent binds to a first attachmentsite of the non-natural molecular scaffold and connects the scaffold toa second binding site of the antigen or antigenic determinant.

3. Compositions, Vaccines, and the Administration Thereof, and Methodsof Treatment

In one embodiment, the invention provides vaccines for the prevention ofinfectious diseases in a wide range of species, particularly mammalianspecies such as human, monkey, cow, dog, cat, horse, pig, etc. Vaccinesmay be designed to treat infections of viral etiology such as HIV,influenza, Herpes, viral hepatitis, Epstein Bar, polio, viralencephalitis, measles, chicken pox, etc.; or infections of bacterialetiology such as pneumonia, tuberculosis, syphilis, etc.; or infectionsof parasitic etiology such as malaria, trypanosomiasis, leishmaniasis,trichomoniasis, amoebiasis, etc.

In another embodiment, the invention provides vaccines for theprevention of cancer in a wide range of species, particularly mammalianspecies such as human, monkey, cow, dog, cat, horse, pig, etc. Vaccinesmay be designed to treat all types of cancer: lymphomas, carcinomas,sarcomas, melanomas, etc.

In another embodiment of the invention, compositions of the inventionmay be used in the design of vaccines for the treatment of allergies.Antibodies of the IgE isotype are important components in allergicreactions. Mast cells bind IgE antibodies on their surface and releasehistamines and other mediators of allergic response upon binding ofspecific antigen to the IgE molecules bound on the mast cell surface.Inhibiting production of IgE antibodies, therefore, is a promisingtarget to protect against allergies. This should be possible byattaining a desired T helper cell response. T helper cell responses canbe divided into type 1 (T_(H)1) and type 2 (T_(H)2) T helper cellresponses (Romagnani, Immunol. Today 18: 263-266 (1997)). T_(H)1 cellssecrete interferon-gamma and other cytokines which trigger B cells toproduce protective IgG antibodies. In contrast, a critical cytokineproduced by T_(H)2 cells is IL-4, which drive B cells to produce IgE. Inmany experimental systems, the development of T_(H)1 and T_(H)2responses is mutually exclusive since T_(H)1 cells suppress theinduction of TH² cells and vice versa. Thus, antigens that trigger astrong T_(H)1 response simultaneously suppress the development of TH²responses and hence the production of IgE antibodies. Interestingly,virtually all viruses induce a T_(H)1 response in the host and fail totrigger the production of IgE antibodies (Coutelier et al., J. Exp. Med.165: 64-69 (1987)). This isotype pattern is not restricted to liveviruses but has also been observed for inactivated or recombinant viralparticles (Lo-Man et al., Eur. J. Immunol. 28: 1401-1407 (1998)). Thus,by using the processes of the invention (e.g., Alpha VaccineTechnology), viral particles can be decorated with various allergens andused for immunization. Due to the resulting “viral structure” of theallergen, a TH1 response will be elicited, “protective” IgG antibodieswill be produced, and the production of IgE antibodies which causeallergic reactions will be prevented. Since the allergen is presented byviral particles which are recognized by a different set of helper Tcells than the allergen itself, it is likely that the allergen-specificIgG antibodies will be induced even in allergic individuals harboringpre-existing TH² cells specific for the allergen. The presence of highconcentrations of IgG antibodies may prevent binding of allergens tomast cell bound IgE, thereby inhibiting the release of histamine. Thus,presence of IgG antibodies may protect from IgE mediated allergicreactions. Typical substances causing allergies include: grass, ragweed,birch or mountain cedar pollens, house dust, mites, animal danders,mold, insect venom or drugs (e.g., penicillin). Thus, immunization ofindividuals with allergen-decorated viral particles should be beneficialnot only before but also after the onset of allergies. Food allergiesare also very common, and immunization of subjects with particlesdecorated with food allergens should be useful for the treatment ofthese allergies.

In another embodiment, the invention relates to the induction ofspecific Th type 2 (Th2) cells. The inventors surprisingly found thatbacterial Pili induce an antibody response dominated by the IgG1 isotypein mice, indicative of a Th2 response. Antigens coupled to Pili alsoinduced a IgG1 response indicating that coupling of antigens to Pili wassufficient for induction of antigen-specific Th2 response. Many chronicdiseases in humans an animals, such as arthritis, colitis, diabetes andmultiple sclerosis are dominated by Th1 response, where T cells secreteIFNγ and other pro-inflammatory cytokines precipitating disease. Bycontrast, Th2 cells secrete I1-4, I1-13 and also I1-10. The lattercytokine is usually associated with immunosuppression and there is goodevidence that specific Th2 cells can suppress chronic diseases, such asarthritis, colitis, diabetes and multiple sclerosis in vivo. Thus,induction of antigen-specific Th2 cells is desirable for the treatmentof such chronic diseases.

It is known that induction of therapeutic self-specific antibodies mayallow treating a variety of diseases. It is, e.g., known that anti-TNFantibodies can ameliorate symptoms in arthritis or colitis andantibodies specific for the Aβ-peptide may remove plaques from the brainof Alzheimers patients. It will usually be beneficial for the patient ifsuch antibodies can be induced in the absence of a pro-inflammatory Th1response. Thus, self antigens coupled to Pili that induce a strongantibody response but no Th1 response may be optimal for suchimmunotherapy.

In a preferred embodiment, the antigen is the amyloid beta peptide(Aβ₁₋₄₂) (DAEFRHDSGYEVHHQKL VFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO:174),or a fragment thereof. The amyloid beta protein is SEQ ID NO:172. Theamyloid beta precursor protein is SEQ ID NO:173.

The amyloid B peptide (Aβ₁₋₄₂) has a central role in the neuropathologyof Alzheimers disease. Region specific, extracellular accumulation of Aβpeptide is accompanied by microgliosis, cytoskeletal changes, dystrophicneuritis and synaptic loss. These pathological alterations are thoughtto be linked to the cognitive decline that defines the disease.

In a mouse model of Alzheimer disease, transgenic animals engineered toproduce Aβ₁₋₄₂ (PDAPP-mice), develop plaques and neuron damage in theirbrains. Recent work has shown immunization of young PDAPP-mice, usingAβ₁₋₄₂, resulted in inhibition of plaque formation and associateddystrophic neuritis (Schenk, D. et al., Nature 400: 173-77 (1999)).

Furthermore immunization of older PDAPP mice that had already developedAD-like neuropathologies, reduced the extent and progression of theneuropathologies. The immunization protocol for these studies was asfollows; peptide was dissolved in aqueous buffer and mixed 1:1 withcomplete Freunds adjuvant (for primary dose) to give a peptideconcentration of 100 μg/dose. Subsequent boosts used incomplete Freundsadjuvant. Mice received 11 immunizations over an 11 month period.Antibodies titres greater than 1:10 000 were achieved and maintained.Hence, immunization may be an effective prophylactic and therapeuticaction against Alzheimer disease.

In another study, peripherally administered antibodies raised againstAβ₄₂, were able to cross the blood-brain barrier, bind Aβ peptide, andinduce clearance of pre-existing amyloid (Bard, F. et al., NatureMedicine 6: 916-19(2000)). This study utilized either polyclonalantibodies raised against Aβ₁₋₄₂, or monoclonal antibodies raisedagainst synthetic fragments derived from different regions of Aβ. Thusinduction of antibodies can be considered as a potential therapeutictreatment for Alzheimer disease.

In another more specific embodiment, the invention is drawn to vaccinecompositions comprising at least one antigen or antigenic determinantencoded by an Influenza viral nucleic acid, and the use of such vaccinecompositions to elicit immune responses. In an even more specificembodiment, the Influenza antigen or antigenic determinant may be an M2protein (e.g., an M2 protein having the amino acids shown in SEQ ID NO:171, GenBank Accession No. P06821, or in SEQ ID NO: 170, PIR AccessionNo. MFIV62, or fragment thereof (e.g., amino acids from about 2 to about24 in SEQ ID NO: 171, the amino acid sequence in SEQ ID NO: 170.Further, influenza antigens or antigenic determinants may be coupled topili or pilus-like structures. Portions of an M2 protein (e.g., an M2protein having the amino acid sequence in SEQ ID NO: 170), as well asother proteins against which an immunological response is sought,suitable for use with the invention may comprise, or alternativelyconsist of, peptides of any number of amino acids in length but willgenerally be at least 6 amino acids in length (e.g., peptides 6, 7, 8,9, 10, 12, 15, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, or 97 amino acids in length).

In an even more specific embodiment, the Influenza antigen or antigenicdeterminant may be an M2 protein (e.g., an M2 protein having the aminoacids shown in SEQ ID NO: 170, GenBank Accession No. P06821, or in SEQID NO: 212, PIR Accession No. MFIV62, or fragment thereof (e.g., aminoacids from about 2 to about 24 in SEQ ID NO: 171, the amino acidsequence in SEQ ID NO: 170). [0236] As would be understood by one ofordinary skill in the art, when compositions of the invention areadministered to an individual, they may be in a composition whichcontains salts, buffers, adjuvants, or other substances which aredesirable for improving the efficacy of the composition. Examples ofmaterials suitable for use in preparing pharmaceutical compositions areprovided in numerous sources including REMINGTON'S PHARMACEUTICALSCIENCES (Osol, A, ed., Mack Publishing Co., (1980)).

Compositions of the invention are said to be “pharmacologicallyacceptable” if their administration can be tolerated by a recipientindividual. Further, the compositions of the invention will beadministered in a “therapeutically effective amount” (i.e., an amountthat produces a desired physiological effect).

The compositions of the present invention may be administered by variousmethods known in the art, but will normally be administered byinjection, infusion, inhalation, oral administration, or other suitablephysical methods. The compositions may alternatively be administeredintramuscularly, intravenously, or subcutaneously. Components ofcompositions for administration include sterile aqueous (e.g.,physiological saline) or non-aqueous solutions and suspensions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Carriers or occlusive dressings can be used to increaseskin permeability and enhance antigen absorption.

The present invention also provides a composition comprising a bacterialpilin polypeptide to which an antigen or antigenic determinant has beenattached by a covalent bond.

The present invention also provides a composition comprising a fragmentof a bacteriophage coat protein to which an antigen or antigenicdeterminant has been attached by a covalent bond.

The present invention also provides a composition comprising (a)non-natural molecular scaffold comprising (i) a core particle selectedfrom the group consisting of (1) a bacterial pilus or pilin protein; and(2) a recombinant form of a bacterial pilus or pilin protein; and (ii)an organizer comprising at least one first attachment site, wherein theorganizer is connected to the core particle by at least one covalentbond; and (b) an antigen or antigenic determinant with at least onesecond attachment site, the second attachment site being selected fromthe group consisting of (i) an attachment site not naturally occurringwith the antigen or antigenic determinant, and (ii) an attachment sitenaturally occurring with the antigen or antigenic determinant, whereinthe second attachment site is capable of association through at leastone non-peptide bond to the first attachment site; and wherein theantigen or antigenic determinant and the scaffold interact through theassociation to form an ordered and repetitive antigen array.

The present invention also provides a composition comprising (a) anon-natural molecular scaffold comprising (i) a core particle selectedfrom the group consisting of: (1) a bacterial pilus; and (2) arecombinant form of a bacterial pilus; and (ii) an organizer comprisingat least one first attachment site, wherein the organizer is connectedto the core particle by at least one covalent bond; and (b) an antigenor antigenic determinant with at least one second attachment site, thesecond attachment site being selected from the group consisting of (i)an attachment site not naturally occurring with the antigen or antigenicdeterminant; and (ii) an attachment site naturally occurring with theantigen or antigenic determinant, wherein the second attachment site iscapable of association through at least one non-peptide bond to thefirst attachment site; and wherein the antigen or antigenic determinantand the scaffold interact through the association to form an ordered andrepetitive antigen array.

The present invention also provides a composition comprising (a) anon-natural molecular scaffold comprising (i) a virus-like particle thatis a dimer or a multimer of a polypeptide comprising amino acids 1-147of SEQ ID NO:158 as core particle; and (ii) an organizer comprising atleast one first attachment site, wherein the organizer is connected tothe core particle by at least one covalent bond; and (b) an antigen orantigenic determinant with at least one second attachment site, thesecond attachment site being selected from the group consisting of (i)an attachment site not naturally occurring with the antigen or antigenicdeterminant; and (ii) an attachment site naturally occurring with theantigen or antigenic determinant, wherein the second attachment site iscapable of association through at least one non-peptide bond to thefirst attachment site; and wherein the antigen or antigenic determinantand the scaffold interact through the association to form an ordered andrepetitive antigen array.

The present invention also provides a pharmaceutical compositioncomprising any of compositions of the present invention, and apharmaceutically acceptable carrier.

The present invention also provides a vaccine composition comprising anyof compositions of the present invention. The vaccine composition mayfurther comprise at least one adjuvant. The present invention alsoprovides a method of immunizing, comprising administering to a subject avaccine composition of the present invention.

The present invention also provides a composition comprising (a) anon-natural molecular scaffold comprising (i) Hepatitis B virus capsidprotein comprising an amino acid sequence selected from the groupconsisting of (1) the amino acid sequence of SEQ ID NO:89, (2) the aminoacid sequence of SEQ ID NO:90 (3) the amino acid sequence of SEQ IDNO:93, (4) the amino acid sequence of SEQ ID NO:98, (5) the amino acidsequence of SEQ ID NO:99, (6) the amino acid sequence of SEQ ID NO:102,(7)the amino acid sequence of SEQ ID NO:104, (8) the amino acid sequenceof SEQ ID NO:105, (9) the amino acid sequence of SEQ ID NO:106, (10) theamino acid sequence of SEQ ID NO:119, (11) the amino acid sequence ofSEQ ID NO:120, (12) the amino acid sequence of SEQ ID NO:123, (13) theamino acid sequence of SEQ ID NO:125, (14) the amino acid sequence ofSEQ ID NO:131, (15) the amino acid sequence of SEQ ID NO:132, (16) theamino acid sequence of SEQ ID NO:134, (17) the amino acid sequence ofSEQ ID NO:157, and (18) the amino acid sequence of SEQ ID NO:158; and(ii) an organizer comprising at least one first attachment site, whereinthe organizer is connected to the core particle by at least one covalentbond; and (b) an antigen or antigenic determinant with at least onesecond attachment site, the second attachment site being selected fromthe group consisting of (i) an attachment site not naturally occurringwith the antigen or antigenic determinant; and (ii) an attachment sitenaturally occurring with the antigen or antigenic determinant, whereinthe second attachment site is capable of association through at leastone non-peptide bond to the first attachment site; and wherein theantigen or antigenic determinant and the scaffold interact through theassociation to form an ordered and repetitive antigen array. Preferably,the organizer is a polypeptide or residue thereof, wherein the secondattachment site is a polypeptide or residue thereof, and wherein thefirst attachment site is a lysine residue and the second attachment siteis a cysteine residue. Preferably, one or more cysteine residues of theHepatitis B virus capsid protein have been either deleted or substitutedwith another amino acid residue. Preferably, the cysteine residuescorresponding to amino acids 48 and 107 in SEQ ID NO:134 have beeneither deleted or substituted with another amino acid residue.

The present invention also provides a composition comprising: (1) anon-natural molecular scaffold comprising (i) a core particle selectedfrom the group consisting of (1) a bacterial pilus, and (2) arecombinant form of a bacterial pilus or pilin protein; and (ii) anorganizer comprising at least one first attachment site, wherein theorganizer is connected to the core particle by at least one covalentbond; and (2) an antigen or antigenic determinant with at least onesecond attachment site, the second attachment site being selected fromthe group consisting of (i) an attachment site not naturally occurringwith the antigen or antigenic determinant, and (ii) an attachment sitenaturally occurring with the antigen or antigenic determinant, whereinthe second attachment site is capable of association through at leastone non-peptide bond to the first attachment site, wherein the antigenor antigenic determinant and the scaffold interact through theassociation to form an ordered and repetitive antigen array, and whereinthe antigen or antigenic determinant is selected from the groupconsisting of an influenza M2 peptide, the GRA2 polypeptide, the DP178cpeptide, the tumor necrosis factor polypeptide, a tumor necrosis factorpeptide, the B2 peptide, the D2 peptide, and the Aβ peptide.

In the compositions and vaccines of the present invention, for acovalent bond between a first and second attachment site, the covalentbond is preferably not a peptide bond.

If a bacterial pilus is present in a composition or vaccine of thepresent invention, the pilus is preferably a Type-1 pilus of Eschericiacoli. More preferably, pilin subunits of the Type-1 pilus comprises theamino acid sequence shown in SEQ ID NO:146. Preferably, the bacterialpilus and the antigen or antigen determinant are attached via either anaturally or non-naturally occurring attachment. Preferably, the firstattachment site will be a lysine residue, while the second attachmentsite will be a cysteine residue present or engineered on the antigen Ifthe attachment comprises interacting leucine zipper polypeptides, thepolypeptides are preferably JUN and/or FOS leucine zipper polypeptides.

In the compositions and vaccines of the present invention that comprisean organizer having a first attachment site, attached to the secondattachment site on the antigen, the organizer is preferably apolypeptide or a residue thereof, and the second attachment site ispreferably a polypeptide or a residue thereof. More preferably, thefirst and/or the second attachment sites comprise an antigen and anantibody or antibody fragment thereto, biotin and avidin, strepavidinand biotin, a receptor and its ligand, a ligand-binding protein and itsligand, interacting leucine zipper polypeptides, an amino group and achemical group reactive thereto, a carboxyl group and a chemical groupreactive thereto, a sulfhydryl group and a chemical group reactivethereto, or a combination thereof More preferably, the first attachmentsite is an amino group, and the second attachment site is a sulfhydrylgroup.

In the compositions and vaccines of the present invention, the antigenis preferably selected from the group consisting of a protein suited toinduce an immune response against cancer cells, a protein suited toinduce an immune response against infectious diseases, a protein suitedto induce an immune response against allergens, and a protein suited toinduce an immune response in farm animals. Preferably, the antigeninduces an immune response against one or more allergens. Morepreferably, the antigen is a recombinant protein of HIV, a recombinantprotein of Influenza virus, a recombinant protein of Hepatitis C virus,a recombinant protein of Toxoplasma, a recombinant protein of Plasmodiumfalciparum, a recombinant protein of Plasmodium vivax, a recombinantprotein of Plasmodium ovale, a recombinant protein of Plasmodiummalariae, a recombinant protein of breast cancer cells, a recombinantprotein of kidney cancer cells, a recombinant protein of prostate cancercells, a recombinant protein of skin cancer cells, a recombinant proteinof brain cancer cells, a recombinant protein of leukemia cells, arecombinant profiling, a recombinant protein of bee sting allergy, arecombinant protein of nut allergy, a recombinant protein of foodallergies, or a recombinant protein of asthma, or a recombinant proteinof Chlamydia.

In the method of immunizing provided by the present invention, theimmunization produces an immune response in the subject. Preferably, theimmunization produces a humoral immune response, a cellular immuneresponse, a humoral and a cellular immune response, or a protectiveimmune response.

In the compositions and vaccines of the present invention, the antigenor antigenic determinant is attached to the non-natural molecularscaffold through the first attachment site, to form an antigen array orantigenic determinant array. Preferably, the array is ordered and/orrepetitive.

In the compositions and vaccines of the present invention, the firstand/or the second attachment sites are preferably attached via either anon-naturally occurring attachment, or by an attachment comprisinginteracting leucine zipper polypeptides. More preferably, theinteracting leucine zipper polypeptides are JUN and/or FOS leucinezipper polypeptides.

The present invention also provides a method for making the compositionsand vaccines of the present invention, comprising combining the antigenor antigenic determinant with the non-natural molecular scaffold throughthe first attachment site and organizer present on the non-naturalmolecular scaffold.

In addition to vaccine technologies, other embodiments of the inventionare drawn to methods of medical treatment for cancer, allergies, andchronic diseases.

Following is a protocol for analyzing pili by SDS-PAGE Analysis. Addtrichloroacetic acid to a final concentration of 10% to the pilisolution containing approx. 50 ug of pili. Vortex and incubate for 10minutes on ice. Centrifuge at maximal speed for 5 minutes in amicrocentrifuge. Discard the supernatant and resuspend the pellet in 50ul of a 8.5 M guanidiniumhydrochloride, pH 3 solution. Heat the samplefor 15 minutes at 70° C. Precipitate the protein by adding 1.5 ml ofEthanol precooled at −20° C., and centrifuge 5 minutes at RT at maximalspeed. Resuspend the pellet in 15 ul of a 10 mM Tris, pH 8 buffer. AddSDS-PAGE sample buffer, vortex shortly and heat the sample 10 minutes at100° C. Load the sample on a 12% gel.

EXAMPLES

Enzymes and reagents used in the experiments that follow included: T4DNA ligase obtained from New England Biolabs; Taq DNA Polymerase,QIAprep Spin Plasmid Kit, QIAGEN Plasmid Midi Kit, QiaExII GelExtraction Kit, QIAquick PCR Purification Kit obtained from QIAGEN;QuickPrep Micro mRNA Purification Kit obtained from Pharmacia;SuperScript One-step RT PCR Kit, fetal calf serum (FCS), bacto-tryptoneand yeast extract obtained from Gibco BRL; Oligonucleotides obtainedfrom Microsynth (Switzerland); restriction endonucleases obtained fromBoehringer Mannheim, New England Biolabs or MBI Fermentas; Pwopolymerase and dNTPs obtained from Boehringer Mannheim. HP-1 medium wasobtained from Cell culture technologies (Glattbrugg, Switzerland). Allstandard chemicals were obtained from Fluka-Sigma-Aldrich, and all cellculture materials were obtained from TPP.

DNA manipulations were carried out using standard techniques. DNA wasprepared according to manufacturer instruction either from a 2 mlbacterial culture using the QIAprep Spin Plasmid Kit or from a 50 mlculture using the QIAGEN Plasmid Midi Kit. For restriction enzymedigestion, DNA was incubated at least 2 hours with the appropriaterestriction enzyme at a concentration of 5-10 units (U) enzyme per mgDNA under manufacturer recommended conditions (buffer and temperature).Digests with more than one enzyme were performed simultaneously ifreaction conditions were appropriate for all enzymes, otherwiseconsecutively. DNA fragments isolated for further manipulations wereseparated by electrophoresis in a 0.7 to 1.5% agarose gel, excised fromthe gel and purified with the QiaExII Gel Extraction Kit according tothe instructions provided by the manufacturer. For ligation of DNAfragments, 100 to 200 pg of purified vector DNA were incubated overnightwith a threefold molar excess of the insert fragment at 16° C. in thepresence of 1 U T4 DNA ligase in the buffer provided by the manufacturer(total volume: 10-20 μl). An aliquot (0. 1 to 0.5 μl) of the ligationreaction was used for transformation of E. coli XL1-Blue (Stratagene).Transformation was done by electroporation using a Gene Pulser (BioRAD)and 0.1 cm Gene Pulser Cuvettes (BioRAD) at 200 Q, 25 pF, 1.7 kV. Afterelectroporation, the cells were incubated with shaking for 1 h in 1 mlS.O.B. medium (Miller, 1972) before plating on selective S.O.B. agar.

Example 1 Insertion of the JUN Amphiphatic Helix Domain Within E2

In the vector pTE5′2J (Hahn et al., Proc. Natl. Acad. Sci. USA 89:2679-2683, (1992)), MluI and a BstEII restriction enzyme sites wereintroduced between codons 71 (Gln) and 74 (Thr) of the structuralprotein E2 coding sequence, resulting in vector pTE5′2JBM. Introductionof these restriction enzymes sites was done by PCR using the followingoligonucleotides: Oligo 1: E2insBstEII/BssHII: (SEQ. ID NO: 1)5′-ggggACGCGTGCAGCAggtaaccaccgTTAAAGAAGGCACC-3′ Oligo 2: E2insMluIStuI:(SEQ ID NO: 2) 5′-cggtggttaccTGCTGCACGCGTTGCTTAAGCGACATGTAGCGG-3′ Oligo3: E2insStuI: (SEQ ID NO: 3) 5′-CCATGAGGCCTACGATACCC-3′ Oligo4:E2insBssHII: (SEQ ID NO: 4) 5′-GGCACTCACGGCGCGCTTTACAGGC-3′

For the PCR reaction, 100 pmol of each oligo was used with 5 ng of thetemplate DNA in a 100 μl reaction mixture containing 4 units of Taq orPwo polymerase, 0.1 mM dNTPs and 1.5 mM MgSO₄. All DNA concentrationswere determined photometrically using the GeneQuant apparatus(Pharmacia). Polymerase was added directly before starting the PCRreaction (starting point was 95° C.). Temperature cycling was done inthe following manner and order: 95° C. for 2 minutes; 5 cycles of 95° C.(45 seconds), 53° C. (60 seconds), 72° C. (80 seconds); and 25 cycles of95° C. (45 seconds), 57° C. (60 seconds), 72° C. (80 seconds).

The two PCR fragments were analyzed and purified by agarosegelelectrophoresis. Assembly PCR of the two PCR fragments using oligo 3and 4 for amplification was carried out to obtain the final construct.

For the assembly PCR reaction, 100 pmol of each oligo was used with 2 ngof the purified PCR fragments in a 100 μl reaction mixture containing 4units of Taq or Pwo polymerase, 0.1 mM dNTPs and 1.5 mM MgSO₄. All DNAconcentrations were determined photometrically using the GeneQuantapparatus (Pharmacia). Polymerase was added directly before starting thePCR reaction (starting point was 95° C.). Temperature cycling was donein the following manner and order: 95° C. for 2 minutes; 5 cycles of 95°C. (45 seconds), 57° C. (60 seconds), 72° C. (90 seconds); and 25 cyclesof 95° C. (45 seconds), 59° C. (60 seconds), 72° C. (90 seconds).

The final PCR product was purified using Qia spin PCR columns (Qiagen)and digested in an appropriate buffer using 10 units each of BssHII andStuI restriction endonucleases for 12 hours at 37° C. The DNA fragmentswere gel-purified and ligated into BssHII/StuI digested and gel-purifiedpTE5′2J vector (Hahn et al., Proc. Natl Acad. Sci. USA 89: 2679-2683).The correct insertion of the PCR product was first analyzed by BstEIIand MluI restriction analysis and then by DNA sequencing of the PCRfragment.

The DNA sequence coding for the JUN amphiphatic helix domain wasPCR-amplified from vector pJuFo (Crameri and Suter, Gene 137: 69 (1993))using the following oligonucleotides: Oligo 5: JUNBstEII: (SEQ ID NO: 5)5′- CCTTCTTTAAcggtggttaccTGCTGGCAACCAACGTGGTTCATGAC-3′ Oligo 6: MluIJUN:(SEQ ID NO: 6) 5′-AAGCATGCTGCacgcgtgTGCGGTGGTCGGATCGCCCGGC-3′

For the PCR reaction, 100 pmol of each oligo was used with 5 ng of thetemplate DNA in a 100 μl reaction mixture containing 4 units of Taq orPwo polymerase, 0.1 mM dNTPs and 1.5 mM MgSO₄. All DNA concentrationswere determined photometrically using the GeneQuant apparatus(Pharmacia). Polymerase was added directly before starting the PCRreaction (starting point was 95° C.). Temperature cycling was done inthe following order and manner: 95° C. for 2 minutes; 5 cycles of 95° C.(45 seconds), 60° C. (30 seconds), 72° C. (25 seconds); and 25 cycles of95° C. (45 seconds), 68° C. (30 seconds), 72° C. (20 seconds).

The final PCR product was gel-purified and ligated into EcoRV digestedand gel-purified pBluescriptII(KS⁻). From the resulting vector, the JUNsequence was isolated by cleavage with MluI/BstEII purified with QiaExIIand ligated into vector pTE5′2JBM (previously cut with the samerestriction enzymes) to obtain the vector pTE5′2J: E2JUN.

Example 2 Production of Viral Particles Containing E2-JUN Using thepCYTts System

The structural proteins were PCR amplified using pTE5′2J: E2JUN astemplate and the oligonucleotides XbalStruct(ctatcaTCTAGAATGAATAGAGGATTCTTTAAC (SEQ ID NO:12)) and StructBsp1201(tcgaatGGGCCCTCATCTTCGTGTGCTAGTCAG (SEQ ID NO:87)). For the PCR 100 pmolof each loligo was used and 5 ng of the template DNA was used in the 100μl reaction mixture, containing 4 units of Tac or Pwo polymerase, 0.1 mMdNTPs and 1.5 mM MgSO₄. All DNA concentrations were determinedphotometrically using the GeneQuant apparatus (Pharmacia). Thepolymerase was added directly before starting the PCR reaction (startingpoint was 95° C.). The temperature cycles were as follows: 95° C. for 3minutes, followed by 5 cycles of 92° C. (30 seconds), 54° C. (35seconds), 72° C. (270 seconds) and followed by 25 cycles of 92° C. (30seconds), 63° C. (35 seconds), 72° C. (270 seconds. The PCR product wasgel purified and digested with the restriction enzymes Xbal/Bsp1201 andligated into vector pCYTts previously cleaved with the same enzymes (WO99/50432)

Twenty μg of pCYTtsE2: JUN were incubated with 30 U of ScaI in anappropriate buffer for at least 4 hours at 37° C. The reaction wasstopped by phenol/chloroform extraction, followed by an isopropanolprecipitation of the linerized DNA. The restriction reaction was checkedby agarose gel eletrophoresis. For the transfection, 5.4 μg oflinearized pCYTtsE2: JUN was mixed with 0.6 μg of linearized pSV2Neo in30 μl H₂O and 30 μl of 1 M CaCl₂ solution were added. After addition of60 μl phosphate buffer (50 mM HEPES, 280 mM NaCl, 1.5 mM Na₂ HPO₄, pH7.05), the solution was vortexed for 5 seconds, followed by anincubation at room temperature for 25 seconds. The solution wasimmediately added to 2 ml HP-1 medium containing 2% FCS (2% FCS medium).The medium of an 80% confluent BHK21 cell culture in a 6-well plate wasthen replaced with the DNA containing medium. After an incubation for 5hours at 37° C. in a CO₂ incubator, the DNA containing medium wasremoved and replaced by 2 ml of 15% glycerol in 2% FCS medium. Theglycerol containing medium was removed after a 30 second incubationphase, and the cells were washed by rinsing with 5 ml of HP-1 mediumcontaining 10% FCS. Finally 2 ml of fresh HP-1 medium containing 10% FCSwas added.

Stably transfected cells were selected and grown in selection medium(HP-1 medium, supplemented with G418) at 37° C. in a CO₂ incubator. Whenthe mixed population was grown to confluency, the culture was split totwo dishes, followed by a 12 hours growth period at 37° C. One dish ofthe cells was shifted to 30° C. to induce the expression of the viralparticles; the other dish was kept at 37° C.

The expression of viral particles was determined by Western blotting(FIG. 1). Culture medium (0.5 ml) was methanol/chloroform precipitated,and the pellet was resuspended in SDS-PAGE sample buffer. Samples wereheated for 5 minutes at 95° C. before being applied to 15% acrylamidegel. After SDS-PAGE, proteins were transferred to Protan nitrocellulosemembranes (Schleicher & Schuell, Germany) as described by Bass and Yang,in Creighton, T. E., ed., Protein Function: A Practical Approach, 2ndEdn., IRL Press, Oxford (1997), pp. 29-55. The membrane was blocked with1% bovine albumin (Sigma) in TBS (10×TBS per liter: 87.7 g NaCl, 66.1 gTrizma hydrochloride (Sigma) and 9.7 g Trizma base (Sigma), pH 7.4) for1 hour at room temperature, followed by an incubation with ananti-E1/E2antibody (polyclonal serum) for 1 hour. The blot was washed 3times for 10 minutes with TBS-T (TBS with 0.05% Tween20), and incubatedfor 1 hour with an alkaline-phosphatase-anti-rabbit IgG conjugate (0.1μg/ml, Amersham Life Science, England). After washing 2 times for 10minutes with TBS-T and 2 times for 10 minutes with TBS, the developmentreaction was carried out using alkaline phosphatase detection reagents(10 ml AP buffer (100 mM Tris/HCl, 100 nM NaCl, pH 9.5) with 50 μl NBTsolution (7.7% Nitro Blue Tetrazolium (Sigma) in 70% dimethylformamide)and 37 μl of X-Phosphate solution (5% of 5-bromo-4-chloro-3-indolylphosphate in dimethylformamide).

The production of viral particles is shown in FIG. 1. The Western Blotpattern showed that E2-JUN (lane 1) migrated to a higher molecularweight in SDS-PAGE compared to wild type E2 (lane 2) and the BHK21 hostcell line did not show any background.

Example 3 Production of Viral Particles Containing E2-JUN Using thepTE5′2JE2: JUN Vector

RNase-free vector (1.0 μg) was linerarized by PvuI digestion.Subsequently, in vitro transcription was carried out using an SP6 invitro transcription kit (InvitroscripCAP by InvitroGen, Invitrogen BV,NV Leek, Netherlands). The resulting 5′-capped mRNA was analyzed on areducing agarose-gel.

In vitro transcribed mRNA (5 μg) was electroporated into BHK 21 cells(ATCC: CCL10) according to Invitrogen's manual (Sindbis Expressionsystem, Invitrogen BV, Netherlands). After 10 hours incubation at 37°C., the FCS containing medium was exchanged by HP-1 medium without FCS,followed by an additional incubation at 37° C. for 10 hours. Thesupernatant was harvested and analyzed by Western blot analysis forproduction of viral particles exactly as described in Example 2.

The obtained result was identical to the one obtained with pCYTtsE2: JUNas shown in FIG. 2.

Example 4 Fusion of Human Growth Hormone (hGH) to the FOS Leucine ZipperDomain (OmpA Signal Sequence)

The hGH gene without the human leader sequence was amplified from theoriginal plasmid (ATCC 31389) by PCR. Oligo 7 with an internal XbaI sitewas designed for annealing at the 5′ end of the hGH gene, and oligo 9with an internal EcoRI site primed at the 3′ end of the hGH gene. Forthe PCR reaction, 100 pmol of each oligo and 5 ng of the template DNAwas used in the 75 μl reaction mixture (4 units of Taq or Pwopolymerase, 0.1 mM dNTPs and 1.5 mM MgSO₄).

PCR cycling was performed in the following manner: 30 cycles with anannealing temperature of 60° C. and an elongation time of 1 minute at72° C.

The gel purified and isolated PCR product was used as a template for asecond PCR reaction to introduce the ompA signal sequence and theShine-Dalgarno sequence. For the PCR reaction, 100 pmol of oligo 8 and 9and 1 ng of the template PCR fragment was used in the 75 μl reactionmixture (4 units of Taq or Pwo polymerase, 0.1 mM dNTPs and 1.5 mMMgSO₄). The annealing temperature for the first five cycles was 55° C.with an elongation time of 60 seconds at 72° C.; another 25 cycles wereperformed with an annealing temperature of 65° C. and an elongation timeof 60 seconds at 72° C.

Oligo7: gggtctagattcccaaccattcccttatccaggctttttgac aacgctatgctccgcgcccatcgtctgcaccagctggcctttgacacc (SEQ ID NO:7); oligo 8:gggtctagaaggaggtaaaaaacgatgaaaaagacagctatcgcgattgcagtggcactggctggtttcgctaccgtagcgcaggccttcccaaccattcccttatcc (SEQ ID NO:8); oligo 9: cccgaattcctagaagccacagctgccctcc(SEQ ID NO:9).

The resulting recombinant hGH gene was subcloned into pBluescript viaXbaI/EcoRI. The correct sequence of both strands was confirmed by DNAsequencing.

The DNA sequence coding for the FOS amphiphatic helix domain wasPCR-amplified from vector pJuFo (Crameri & Suter Gene 137: 69 (1993))using the oligonucleotides: omp-FOS: (SEQ ID NO: 10) 5′-ccTGCGGTGGTCTGACCGACACCC-3′ FOS-hgh: (SEQ ID NO: 11) 5′-ccgcggaagagccaccGCAACCACCGTGTGCCGCCAGGATG-3′

For the PCR reaction, 100 pmol of each oligo and 5 ng of the templateDNA was used in the 75 μl reaction mixture (4 units of Taq or Pwopolymerase, 0.1 mM dNTPs and 1.5 mM MgSO₄). The temperature cycles wereas follows:

95° C. for 2 minutes, followed by 5 cycles of 95° C. (45 seconds), 60°C. (30 seconds), 72° C. (25 seconds) and followed by 25 cycles of 95° C.(45 seconds), 68° C. (30 seconds), 72° C. (20 seconds).

The PCR product was purified, isolated and cloned into the StuI digestedpBluescript-ompA-hGH. The hybrid gene was then cloned into the pKK223-3Plasmid (Pharmacia).

Example 5 Bacterial Expression of FOS-hGH

The ompA-FOS-hGH in pkk223-3 was expressed under the control of theinducible IPTG-dependend promoter using JM101 as E. coli host strain.Expression was performed in shaker flask. Cells were induced with 1 mMIPTG (final concentration) at an OD600 of 0.5. Expression was continuedfor 10 hours at 37° C. Cells were harvested by centrifugation at 3600 at10° C. for 15 min. The cell pellet was frozen (−20° C. or liq. N₂) andstored for 16 hours. The pellet was then thawed at 4° C. and resuspendedin 10 ml 10 mM Tris-HCl, pH 7.4 containing 600 mM sucrose. Afterstirring for 15 min at 4° C., periplasmic proteins were released by anosmotic shock procedure. Chilled (4° C.) deionized H₂O was added, andthe suspension was stirred for 30 min at 4° C. The sludge was diluted,resuspended, and lysozyme was added to degrade the cell wall of thebacteria. The cells and the periplasmic fraction spheroplasts wereseparated by centrifugation for 20 min at 11000×g at 4° C. TheFOS-hGH-containing supernatant was analyzed by reducing and non-reducingSDS-Page and Dot Blot. Dot Blot was carried out as described in Example8, using an anti-hGH antibody (Sigma) as the first antibody and analkaline phosphatase (AP)-anti-mouse antibody conjugate as the secondantibody.

Full length, correctly processed FOS-hGH could be detected underreducing and non-reducing conditions. Part of FOS-hGH was bound toother, non-identified proteins due to the free cysteines present in theFOS amphiphatic helix. However, more than 50% of expressed FOS-hGHoccurred in its native monomeric conformation (FIG. 3).

Purified FOS-hGH will be used to perform first doping experiments withJUN containing viral particles.

Example 6 Construction of the pAV Vector Series for Expression of FOSFusion Proteins

A versatile vector system was constructed that allowed either cytplasmicproduction or secretion of N- or C-terminal FOS fusion proteins in E.coli or production of N- or C-terminal FOS fusion proteins in eukaryoticcells. The vectors pAV1-pAV4 which was designed for production of FOSfusion proteins in E. coli, encompasses the DNA cassettes listed below,which contain the following genetic elements arranged in differentorders: (a) a strong ribosome binding site and 5′-untranslated regionderived from the E. coli ompA gene (aggaggtaaaaaacg) (SEQ ID NO:13); (b)a sequence encoding the signal peptide of E. coli outer membrane proteinOmpA (MKKTAIAIAVALAGFATVAQA) (SEQ ID NO:14); (c) a sequence coding forthe FOS dimerization domain flanked on both sides by two glycineresidues and a cystein residue(CGGLTDTLQAETDQVEDEKSALQTEIANLLKEKEKLEFILAAHGGC) (SEQ ID NO:15); and (d)a region encoding a short peptidic linker (AAASGG (SEQ ID NO:16) orGGSAAA (SEQ ID NO:17)) connecting the protein of interest to the FOSdimerization domain. Relevant coding regions are given in upper caseletters. The arrangement of restriction cleavage sites allows easyconstruction of FOS fusion genes with or without a signal sequence. Thecassettes are cloned into the EcoRI/HindIII restriction sites ofexpression vector pKK223-3 (Pharmacia) for expression of the fusiongenes under control of the strong tac promotor.

pAV1

This vector was designed for the secretion of fusion proteins with FOSat the C-terminus into the E. coli periplasmic space. The gene ofinterest (g.o.i.) may be ligated into the StuI/NotI sites of the vector.EcoRI        31/11 gaa ttc agg agg taa aaa acg ATG AAA AAG ACA GCT ATCGCG ATT GCA GTG GCA CTG GCT                             M   K   K   T   A                             I   A   I   A  V   A   L   A61/21                    StuI               NotI GGT TTC GCT ACC GTA GCGCAG GCC tgg gtg ggg GCG GCC GCT TCT GGT GGT TGC GGT GGT G   F   A   T   V   A   Q   A    (goi)      A  A   A   S   G G   C   G   G 121/41                                  151/51 CTG ACCGAC ACC CTG CAG GCG GAA ACC GAC CAG GTG GAA GAC GAA AAA TCC GCG CTG CAA L   T   D   T   L   Q   A   E   T   D   Q   V  E   D   E   K S   A   L   Q 181/61                                  211/71 ACC GAAATC GCG AAC CTG CTG AAA GAA AAA GAA AAG CTG GAG TTC ATC CTG GCG GCA CAC T   E   I   A   N   L   L   K   E   K   E   K  L   E   F   I L   A   A   H 241/81       HindIII GGT GGT TGC taa gct t (SEQ ID NO:18)  G   G   C   *   A (SEQ ID NOs: 14 and 19)pAV2

This vector was designed for the secretion of fusion proteins with FOSat the N-terminus into the E. coli periplasmic space. The gene ofinterest (g.o.i.) ligated into the NotI/EcoRV (or NotI/HindIII) sites ofthe vector. EcoRI                                   31/11 gaa ttc aggagg taa aaa acg ATG AAA AAG ACA GCT ATC GCG ATT GCA GTG GCA CTG GCT                             M   K   K   T   A                             I   A   I   A     V   A   L   A61/21                   StuI            91/31 GGT TTC GCT ACC GTA GCGCAG GCC TGC GGT GGT CTG ACC GAC ACC CTG CAG GCG GAA ACC G   F   A   T   V   A   Q   A   C   G   G   L  T   D   T   L Q   A   E   T 121/41                                  151/51 GAC CAGGTG GAA GAC GAA AAA TCC GCG CTG CAA ACC GAA ATC GCG AAC CTG CTG AAA GAA D   Q   V   E   D   E   K   S   A   L   Q   T  E   I   A   N L   L   K   E 181/61                                  211/71    NotIAAA GAA AAG CTG GAG TTC ATC CTG GCG GCA CAC GGT GGT TGC GGT GGT TCT GCGGCC GCT  K   E   K   L   E   F   I   L   A   A   H   G  G   C   G   G S   A   A   A 241/81      EcoRV   HindIII ggg tgt ggg gat atc aag ctt(SEQ ID NO: 20)   (goi) (SEQ ID NO: 21)pAV3

This vector was designed for the cytoplasmic production of fusionproteins with FOS at the C-terminus in E. coli. The gene of interest(g.o.i.) may be ligated into the EcoRV/NotI sites of the vector.EcoRI                   EcoRV               NotI gaa ttc agg agg taa aaagat atc ggg tgt ggg GCG GCC GCT TCT GGT GGT TGC GGT GGT                                   (goi)     A   A                                             A   S                                             G  G   C   G   G61/21                                   91/31 CTG ACC GAC ACC CTG CAGGCG GAA ACC GAC CAG GTG GAA GAC GAA AAA TCC GCG CTG CAA L   T   D   T   L   Q   A   E   T   D   Q   V  E   D   E   K S   A   L   Q 121/41                                  151/51 ACC GAAATC GCG AAC CTG CTG AAA GAA AAA GAA AAG CTG GAG TTC ATC CTG GCG GCA CAC T   E   I   A   N   L   L   K   E   K   E   K  L   E   F   I L   A   A   H 181/61       HindIII GGT GGT TGC taa gct t (SEQ ID NO:22)  G   G   C   * (SEQ ID NO: 23)pAV4

This vector is designed for the cytoplasmic production of fusionproteins with FOS at the N-terminus in E. coli. The gene of interest(g.o.i.) may be ligated into the NotI/EcoRV (or NotI/HindIII) sites ofthe vector. The N-terminal methionine residue is proteolytically removedupon protein synthesis (Hirel et al., Proc. Natl. Acad. Sci. USA 86:8247-8251 (1989)). EcoRI                                   31/11 gaa ttcagg agg taa aaa acg ATG GCT TGC GGT GGT CTG ACC GAC ACC CTG CAG GCG GAA E   F   R   R   *   K   T   M   A   C   G   G  L   T   D   T L   Q   A   E 61/21                                   91/31 ACC GAC CAGGTG GAA GAC GAA AAA TCC GCG CTG CAA ACC GAA ATC GCG AAC CTG CTG AAA T   D   Q   V   E   D   E   K   S   A   L   Q  T   E   I   A N   L   L   K 121/41                                  151/51       NotI GAA AAA GAA AAG CTG GAG TTC ATC CTG GCG GCA CAC GGT GGT TGCGGT GGT TCT GCG GCC  E   K   E   K   L   E   F   I   L   A   A   H G   G   C   G  G   S   A   A 181/61          EcoRV   HindIII GCT gggtgt ggg gat atc aag ctt (SEQ ID NO: 24) A     (goi) (SEQ ID NOs: 88 and25)

The vectors pAV5 and pAV6, which are designed for eukaryotic productionof FOS fusion proteins, encompasses the following genetic elementsarranged in different orders: (a) a region coding for the leader peptideof human growth hormone (MATGSRTSLLLAFGLLCLPWLQEGSA) (SEQ ID NO:26); (b)a sequence coding for the FOS dimerization domain flanked on both sidesby two glycine residues and a cysteine residue(CGGLTDTLQAETDQVEDEKSALQTEIANLLKEKEKLEFILAAHGGC) (SEQ ID NO:15); and (c)a region encoding a short peptidic linker (AAASGG (SEQ ID NO:16) orGGSAAA (SEQ ID NO:17)) connecting the protein of interest to the FOSdimerization domain. Relevant coding regions are given in upper caseletters. The arrangement of restriction cleavage sites allows easyconstruction of FOS fusion genes. The cassettes are cloned into theEcoRI/HindIII restriction sites of the expression vector pMPSVEH (Arteltet al., Gene 68: 213-219 (1988)).

pAV5

This vector is designed for the eukaryotic production of fusion proteinswith FOS at the C-terminus. The gene of interest (g.o.i.) may beinserted between the sequences coding for the hGH signal sequence andthe FOS domain by ligation into the Eco47III/NotI sites of the vector.Alternatively, a gene containing its own signal sequence may be fused tothe FOS coding region by ligation into the StuI/NotI sites.EcoRI   StuI                            31/11 gaa ttc agg cct ATG GCTACA GGC TCC CGG ACG TCC CTG CTC CTG GCT TTT GGC CTG CTC                 M   A   T   G   S   R   T   S   L   L   L   A F   G   L   L 61/21                           Eco47III            NotITGC CTG CCC TGG CTT CAA GAG GGC AGC GCT ggg tgt ggg GCG GCC GCT TCT GGTGGT TGC  C   L   P   W   L   Q   E   G   S   A     (goi)     A   A   A S   G   G   C 121/41                                  151/51 GGT GGTCTG ACC GAC ACC CTG CAG GCG GAA ACC GAC CAG GTG GAA GAC GAA AAA TCC GCG G   G   L   T   D   T   L   Q   A   E   T   D   Q   V   E   D E   K   S   A 181/61                                  211/71 CTG CAAACC GAA ATC GCG AAC CTG CTG AAA GAA AAA GAA AAG CTG GAG TTC ATC CTG GCG L   Q   T   E   I   A   N   L   L   K   E   K   E   K   L   E F   I   L   A 241/81               HindIII GCA CAC GGT GGT TGCtaa gct t (SEQ ID NO: 27)  A   H   G   G   C   * (SEQ ID NO: 28)pAV6

This vector is designed for the eukaryotic production of fusion proteinswith FOS at the N-terminus. The gene of interest (g.o.i.) may be ligatedinto the NotI/StuI (or NotI/HindIII) sites of the vector.EcoRI                                   31/11 gaa ttc ATG GCT ACA GGCTCC CGG ACG TCC CTG CTC CTG GCT TTT GGC CTG CTC TGC CTG         M   A   T   G   S   R   T   S   L   L          L   A   F   G L   L   C   L 61/21                   Eco47III        91/31 CCC TGG CTTCAA GAG GGC AGC GCT TGC GGT GGT CTG ACC GAC ACC CTG CAG GCG GAA ACC P   W   L   Q   E   G   S   A   C   G   G   L  T   D   T   L Q   A   E   T 121/41                                  151/51 GAC CAGGTG GAA GAC GAA AAA TCC GCG CTG CAA ACC GAA ATC GCG AAC CTG CTG AAA GAA D   Q   V   E   D   E   K   S   A   L   Q   T  E   I   A   N L   L   K   E 181/61                                  211/71 NotI AAAGAA AAG CTG GAG TTC ATC CTG GCG GCA CAC GGT GGT TGC GGT GGT TCTGCG GCC GCT  K   E   K   L   E   F   I   L   A   A   H   G G   C   G   G  S   A   A   A 241/81      StuI    HindIII ggg tgt gggagg cct aag ctt (SEQ ID NO: 29)   (goi) (SEQ ID NO: 30)Construction of Expression Vectors pAV1-pAV6

The following oligonucleotides have been synthesized for construction ofexpression vectors pAV1-pAV6: FOS-FOR1: (SEQ ID NO: 31)CCTGGGTGGGGGCGGCCGCTTCTGGTGGTTGCGGTGGTCTGACC; FOS-FOR2: (SEQ ID NO: 32)GGTGGGAATTCAGGAGGTAAAAAGATATCGGGTGTGGGGCGGCC; FOS-FOR3: (SEQ ID NO: 33)GGTGGGAATTCAGGAGGTAAAAAACGATGGCTTGCGGTGGTCTGACC; FOS-FOR4: (SEQ ID NO:34) GCTTGCGGTGGTCTGACC; FOS-REV1: (SEQ ID NO: 35)CCACCAAGCTTAGCAACCACCGTGTGC; FOS-REV2: (SEQ ID NO: 36)CCACCAAGCTTGATATCCCCACACCCAGCGGCCGCAGAACCACCGC AACCACCG; FOS-REV3: (SEQID NO: 37) CCACCAAGCTTAGGCCTCCCACACCCAGCGGC; OmpA-FOR1: (SEQ ID NO: 38)GGTGGGAATTCAGGAGGTAAAAAACGATG; hGH-FOR1: (SEQ ID NO: 39)GGTGGGAATTCAGGCCTATGGCTACAGGCTCC; and hGH-FOR2: (SEQ ID NO: 40)GGTGGGAATTCATGGCTACAGGCTCCC.

For the construction of vector pAV2, the regions coding for the OmpAsignal sequence and the FOS domain were amplified from the ompA-FOS-hGHfusion gene in vector pKK223-3 (see Example 5) using the primer pairOmpA-FOR1/FOS-REV2. The PCR product was digested with EcoRI/HindIII andligated into the same sites of vector pKK223-3 (Pharmacia).

For the construction of vector pAV1, the FOS coding region was amplifiedfrom the ompA-FOS-hGH fusion gene in vector pKK223-3 (see Example 5)using the primer pair FOS-FOR1/FOS-REV1. The PCR product was digestedwith HindIII and ligated into StuI/HindIII digested vector pAV2.

For the construction of vector pAV3, the region coding for the FOSdomain was amplified from vector pAV1 using the primer pairFOS-FOR2/FOS-REV1. The PCR product was digested with EcoRI/HindIII andligated into the same sites of the vector pKK223-3 (Pharmacia).

For the construction of vector pAV4, the region coding for the FOSdomain was amplified from the ompA-FOS-hGH fusion gene in vectorpKK223-3 (see Example 5) using the primer pair FOS-FOR3/FOS-REV2. ThePCR product was digested with EcoRI/HindIII and ligated into the samesites of the vector pKK223-3 (Pharmacia).

For the construction of vector pAV5, the region coding for the hGHsignal sequence is amplified from the hGH-FOS-hGH fusion gene in vectorpSINrepS (see Example 7) using the primer pair hGH-FOR1/hGHREV1. The PCRproduct is digested with EcoRI/NotI and ligated into the same sites ofthe vector pAV1 The resulting cassette encoding the hGH signal sequenceand the FOS domain is then isolated by EcoRI/HindIII digestion andcloned into vector pMPSVEH (Artelt et al., Gene 68: 213-219 (1988))digested with the same enzymes.

For the construction of vector pAV6, the FOS coding region is amplifiedfrom vector pAV2 using the primer pair FOS-FOR4/FOSREV3. The PCR productis digested with HindIII and cloned into Eco47III/HindIII cleaved vectorpAV5. The entire cassette encoding the hGH signal sequence and the FOSdomain is then reamplified from the resulting vector using the primerpair hGH-FOR2/FOSREV3, cleaved with EcoRI/HindIII and ligated intovector pMPSVEH (Artelt et al., Gene 68: 213-219 (1988)) cleaved with thesame enzymes.

Example 7 Construction of FOS-hGH with Human (hGH) Signal Sequence

For eukaryotic expression of the FOS-hGH fusion protein, theOmpA-FOS-hGH fusion gene was isolated from pBluescript: : OmpA-FOS-hGH(see Example 4) by digestion with XbaI/Bsp1201 and cloned into vectorpSINrepS (Invitrogen) cleaved with the same enzymes. The hGH signalsequence was synthesized by PCR (reaction mix: 50 pmol of each primer,dATP, dGTP, dTTP, dCTP (200 μM each), 2.5 U Taq DNA polymerase (Qiagen),50 μl total volume in the buffer supplied by the manufacturer;amplification: 92° C. for 30 seconds, 55° C. for 30 seconds, 72° C. for30 seconds, 30 cycles) using the overlapping oligonucleotidesSig-hGH-FOR (GGGTCTAGAATGGCTACAGGCTCCCGGACGTCCCTGCTCCTGGCTTTTGGCCTGCTCTG) (SEQ ID NO:41) and Sig-hGH-REV(CGCAGGCCTCGGCACTGCCCTCTTGAAGCCAGGGCAGGCAGAGCA GGCCAAAAGCCAG) (SEQ IDNO:42). The PCR product was purified using the QiaExII Kit, digestedwith StuI/XbaI and ligated into vector pSINrep5: : OmpA-FOS-hGH cleavedwith the same enzymes.

Example 8 Eukaryotic Expression of FOS-hGH

RNase-free vector (1.0 μg) (pSINrep5::OmpA-FOS-hGH) and 1.0 μg of DHEB(Bredenbeek et al., J. Virol. 67: 6439-6446 (1993)) were linerarized byScal restriction digest. Subsequently, in vitro transcription wascarried out using an SP6 in vitro transcription kit (InvitroscripCAP byInvitroGen, Invitrogen BV, NV Leek, Netherlands). The resulting5′-capped mRNA was analyzed on reducing agarose-gel.

In vitro, transcribed mRNA 5 μg was electroporated into BHK 21 cells(ATCC: CCL10) according to Invitrogen's manual (Sindbis Expressionsystem, Invitrogen BV, Netherlands). After 10 hours incubation at 37° C.the FCS containing medium was exchanged by P-1 medium without FCS,followed by an additional incubation at 37° C. for 10 hours. Thesupernatant was harvested and analyzed by dot-blot analysis forproduction of FOS-hgh.

Culture media (2.5 μl) was spotted on a nitrocellulose membrane anddried for 10 minutes at room temperature. The membrane was blocked with1% bovine albumin (Sigma) in TBS (10×TBS per liter: 87.7 g NaCl, 66.1 gTrizma hydrochloride (Sigma) and 9.7 g Trizma base (Sigma), pH 7.4) for1 hour at room temperature, followed by an incubation with 2 μg rabbitanti-human hGH antibody (Sigma) in 10 ml TBS-T (TBS with 0.05% Tween20)for 1 hour. The blot was washed 3 times for 10 minutes with TBS-T andincubated for 1 hour with alkaline phosphatase conjugated anti-rabbitIgG (Jackson ImmunoResearch Laboratories, Inc.) diluted 1:5000 in TBS-T.After washing 2 times for 10 minutes with TBS-T and 2 times for 10minutes with TBS, the blot was developed by AP staining as described inExample 2. Results are shown in FIG. 3.

Example 9 Construction of FOS-PLA (N- and C-Terminal)

The following gene is constructed by chemical gene synthesis coding fora catalytically inactive variant (Forster et al., J. Allergy Clin.Immunol. 95: 1229-1235 (1995)) of bee venom phospholipase A₂ (PLA).1/1                                     31/11 ATC ATC TAC CCA GGT ACTCTG TGG TGT GGT CAC GGC AAC AAA TCT TCT GGT CCG AAC GAA I   I   Y   P   G   T   L   W   C   G   H   G  N   K   S   S G   P   N   E 61/21                                   91/31 CTC GGC CGCTTT AAA CAC ACC GAC GCA TGC TGT CGC ACC CAG GAC ATG TGT CCG GAC GTC L   G   R   F   K   H   T   D   A   C   C   R  T   Q   D   M C   P   D   V 121/41                                  151/51 ATG TCTGCT GGT GAA TCT AAA CAC GGG TTA ACT AAC ACC GCT TCT CAC ACG CGT CTC AGC M   S   A   G   E   S   K   H   G   L   T   N  T   A   S   H T   R   L   S 181/61                                  211/71 TGC GACTGC GAC GAC AAA TTC TAC GAC TGC CTT AAG AAC TCC GCC GAT ACC ATC TCT TCT C   D   C   D   D   K   F   Y   D   C   L   K  N   S   A   D T   I   S   S 241/81                                  271/91 TAC TTCGTT GGT AAA ATG TAT TTC AAC CTG ATC GAT ACC AAA TGT TAC AAA CTG GAA CAC Y   F   V   G   K   M   Y   F   N   L   I   D  T   K   C   Y K   L   E   H 301/101                                 331/111 CCG GTAACC GGC TGC GGC GAA CGT ACC GAA GGT CGC TGC CTG CAC TAC ACC GTT GAC AAA P   V   T   G   C   G   E   R   T   E   G   R  C   L   H   Y T   V   D   K 361/121                                 391/131 (SEQ IDNO: 43) TCT AAA CCG AAA GTT TAC CAG TGG TTC GAC CTG CGC AAA TAC (SEQ IDNO: 44)  S   K   P   K   V   Y   Q   W   F   D   L   R  K   Y

For fusion of PLA to the N-terminus of the FOS dimerization domain, theregion is amplified using the oligonucleotides PLA-FOR1(CCATCATCTACCCAGGTAC) (SEQ ID NO:45) and PLA-REV1(CCCACACCCAGCGGCCGCGTATTTGCGCAGGTCG) (SEQ ID NO:46). The PCR product iscleaved with NotI and ligated into vector pAV1 previously cleaved withthe restriction enzymes StuL/NotI. For fusion of PLA to the C-terminusof the FOS dimerization domain, the region is amplified using theoligonucleotides PLA-FOR2 (CGGTGGTTCTGCGGCCGCTATCATCTACCCAGGTAC) (SEQ IDNO : 47) and PLA-REV2 (TTAGTATTTGCGCAGGTCG) (SEQ ID NO:48). The PCRproduct is cleaved with NotI and ligated into vector pAV2 previouslycleaved with the restriction enzymes NotI/EcoRV.

Example 10 Construction of FOS-Ovalbumin Fusion Gene (N- and C-Terminal)

For cloning of the ovalbumin coding sequence, mRNA from chicken oviducttissue is prepared using the QuickPrep™ Micro mRNA Purification Kit(Pharmacia) according to manufacturer instructions. Using theSuperScript™ One-step RT PCR Kit (Gibco BRL), a cDNA encoding the maturepart of ovalbumin (corresponding to nucleotides 68-1222 of the mRNA(McReynolds et al., Nature 273: 723-728 (1978)) is synthesized using theprimers Ova-FOR1 (CCGGCTCCATCGGTGCAG) (SEQ ID NO:49) and Ova-REV1(ACCACCAGAAGCGGCCGCAGGGGAAACACATCTGCC) (SEQ ID NO:50). The PCR productis digested with NotI and cloned into StuI/NotI digested vector pAV1 forexpression of the fusion protein with the FOS dimerization domain at theC terminus. For production of a fusion protein with the FOS dimerizationdomain at the N terminus, the Ovalbumin coding region is amplified fromthe constructed vector (pAV1::Ova) using the primers Ova-FOR2(CGGTGGTTCTGCGGCCGCTGGCTCCATCGGTGCAG) (SEQ ID NO : 51) and Ova-REV2(TTAAGGGGAAACACATCTGCC) (SEQ ID NO:52). The PCR product is digested withNotI and cloned into the NotI/EcoRV digested vector pAV2. Clonedfragments are verified by DNA sequence analysis.

Example 11 Production and Purification of FOS-PLA and FOS OvalbuminFusion Proteins

For cytoplasmic production of FOS fusion proteins, an appropriate E.coli strain was transformed with the vectors pAV3::PLA, pAV4::PLA,pAV3::Ova or pAV4::Ova. The culture was incubated in rich medium in thepresence of ampicillin at 37° C. with shaking. At an optical density(550 nm) of 1, 1 mM IPTG was added and incubation was continued foranother 5 hours. The cells were harvested by centrifugation, resuspendedin an appropriate buffer (e.g., tris-HCl, pH 7.2, 150 mM NaCl)containing DNase, RNase and lysozyme, and disrupted by passage through afrench pressure cell. After centrifugation (Sorvall RC-5C, SS34 rotor,15000 rpm, 10 min, 4° C.), the pellet was resuspended in 25 ml inclusionbody wash buffer (20 mM tris-HCl, 23% sucrose, 0.5% Triton X-100, 1 mMEDTA, pH8) at 4° C. and recentrifuged as described above. This procedurewas repeated until the supernatant after centrifugation was essentiallyclear. Inclusion bodies were resuspended in 20 ml solubilization buffer(5.5 M guanidinium hydrochloride, 25 mM tris-HCl, pH 7.5) at roomtemperature and insoluble material was removed by centrifugation andsubsequent passage of the supernatant through a sterile filter (0.45μm). The protein solution was kept at 4° C. for at least 10 hours in thepresence of 10 mM EDTA and 100 mM DTT and then dialyzed three timesagainst 10 volumes of 5.5 M guanidinium hydrochloride, 25 mM tris-HCl,10 mM EDTA, pH 6. The solution was dialyzed twice against 5 liters of 2M urea, 4 mM EDTA, 0.1 M NH₄Cl, 20 mM sodium borate (pH 8.3) in thepresence of an appropriate redox shuffle (oxidized glutathione/reducedglutathione; cystine/cysteine). The refolded protein was then applied toan ion exchange chromatography. The protein was stored in an appropriatebuffer with a pH above 7 in the presence of 2-10 mM DTT to keep thecysteine residues flanking the FOS domain in a reduced form. Prior tocoupling of the protein with the alphavirus particles, DTT was removedby passage of the protein solution through a Sephadex G-25 gelfiltration column.

Example 12 Constructions of gp140-FOS

The gp140 gene (Swiss-Prot: P03375) without the internal proteasecleavage site was amplified by PCR from the original plasmid pAbT4674(ATCC 40829) containing the full length gp160 gene using the followingoligonucleotides: HIV-1: 5′-ACTAGTCTAGAatgagagtgaaggagaaatatc-3′; (SEQID NO: 53) HIV-end 5′-TAGCATGCTAGCACCGAAtttatctaattccaataattcttg-3′;(SEQ ID NO: 54) HIV-Cleav:5′-gtagcacccaccaaggcaaagCTGAAAGCTACCCAGCTCGAGAAACTGgca-3′; (SEQ ID NO:55) and HIV-Cleav2:5′-caaagctcctattcccactgcCAGTTTCTCGAGCTGGGTAGCTTTCAG-3′. (SEQ ID NO: 56)

For PCR I, 100 pmol of oligo HIV-1 and HIV-Cleav2 and 5 ng of thetemplate DNA were used in the 75 μl reaction mixture (4 units of Taq orPwo polymerase, 0.1 mM dNTPs and 1.5 mM MgSO₄). PCR cycling was done inthe following manner: 30 cycles with an annealing temperature of 60° C.and an elongation time of 2 minutes at 72° C.

For PCR II, 100 pmol of oligo HIV-end and HIV-Cleav and 5 ng of thetemplate DNA were used in the 75 μl reaction mixture, (4 units of Taq orPwo polymerase, 0.1 mM dNTPs and 1.5 mM MgSO₄). PCR cycling was done inthe following manner: 30 cycles with an annealing temperature of 60° C.and an elongation time of 50 seconds at 72° C.

Both PCR fragments were purified, isolated and used in an assembly PCRreaction. For the assembly PCR reaction, 100 pmol of oligo HIV-1 andHIV-end and 2 ng of each PCR fragment (PCRI and PCR II) were used in the75 μl (4 units of Taq or Pwo polymerase, 0.1 mM dNTPs and 1.5 mM MgSO₄).PCR cycling was done in the following manner: 30 cycles with anannealing temperature of 60° C. and an elongation time of 2.5 minutes at72° C. The assembly PCR product was digested XbaI and nheI. The FOSamphiphatic helix was fused in frame to the C-terminal end of gp-140.

The DNA sequence coding for the FOS amphiphatic helix domain wasPCR-amplified from vector pJuFo (Crameri & Suter Gene 137: 69 (1993))using the oligonucleotides: FOS-HIV: (SEQ ID NO: 57)5′-ttcggtgctagcggtggcTGCGGTGGTCTGACCGAC-3′; and FOS-Apa: (SEQ ID NO: 58)5′-gatgctgggcccttaaccGCAACCACCGTGTGCCGCC-3′.

For the PCR reaction, 100 pmol of each oligo and 5 ng of the templateDNA was used in the 75 μl reaction mixture (4 units of Taq or Pwopolymerase, 0.1 mM dNTPs and 1.5 mM MgSO₄). Temperature cycling was doneas follows: 95° C. for 2 minutes, followed by 5 cycles of 95° C. (45seconds), 60° C. (30 seconds), 72° C. (25 seconds) and followed by 25cycles of 95° C. (45 seconds), 68° C. (30 seconds), 72° C. (20 seconds).The obtained PCR fragment was digested with NheI and Bsp120L.

The final expression vector for GP140-FOS was obtained in a 3 fragmentligation of both PCR fragments into pSinRepS. The resultant vectorpSinRep5-GP140-FOS was evaluated by restriction analysis and DNAsequencing.

GP 140-FOS was also cloned into pCYTts via XbaI and Bsp120L to obtain astable, inducible GP140-FOS expressing cell line.

Example 13 Expression of GP140FOS using pSinRepS-GP140FOS

RNase-free vector (1.0 μg) (pSinRep5-GP140-FOS) and 1.0 μg of DHEB(Bredenbeek et al., J. Virol. 67: 6439-6446 (1993)) were linearized byrestriction digestion. Subsequently, in vitro transcription was carriedout using an SP6 in vitro transcription kit (InvitroscripCAP byInvitroGen, Invitrogen BV, NV Leek, Netherlands). The resulting5′-capped mRNA was analyzed on a reducing agarose-gel.

In vitro transcribed mRNA (5 μg) was electroporated into BHK 21 cells(ATCC: CCL1O) according to Invitrogen's manual (Sindbis ExpressionSystem, Invitrogen BV, Netherlands). After 10 hours incubation at 37°C., the FCS containing medium was exchanged by HP-1 medium without FCS,followed by an additional incubation at 37° C. for 10 hours. Thesupernatant was harvested and analyzed by Western blot analysis forproduction of soluble GP140-FOS exactly as described in Example 2.

Example 14 Expression of GP 140FOS Using pCYTts-GP140FOS

pCYT-GP 140-FOS 20 μg was linearized by restriction digestion. Thereaction was stopped by phenol/chloroform extraction, followed by anisopropanol precipitation of the linearized DNA. The restrictiondigestion was evaluated by agarose gel eletrophoresis. For thetransfection, 5.4 μg of linearized pCYTtsGP140-FOS was mixed with 0.6 μgof linearized pSV2Neo in 30 μl H₂O and 30 μl of 1 M CaCl₂ solution wasadded. After addition of 60 μl phosphate buffer (50 mM HEPES, 280 mMNaCl, 1.5 mM Na₂ HPO₄, pH 7.05), the solution was vortexed for 5seconds, followed by an incubation at room temperature for 25 seconds.The solution was immediately added to 2 ml HP-1 medium containing 2% FCS(2% FCS medium). The medium of an 80% confluent BHK21 cell culture(6-well plate) was then replaced by the DNA containing medium. After anincubation for 5 hours at 37° C. in a CO₂ incubator, the DNA containingmedium was removed and replaced by 2 ml of 15% glycerol in 2% FCSmedium. The glycerol containing medium was removed after a 30 secondincubation phase, and the cells were washed by rinsing with 5 ml of HP-1medium containing 10% FCS. Finally 2 ml of fresh HP-1 medium containing10% FCS was added.

Stably transfected cells were selected and grown in selection medium(HP-1 medium supplemented with G418) at 37° C. in a CO₂ incubator. Whenthe mixed population was grown to confluency, the culture was split totwo dishes, followed by a 12 h growth period at 37° C. One dish of thecells was shifted to 30° C. to induce the expression of solubleGP140-FOS. The other dish was kept at 37° C.

The expression of soluble GP140-FOS was determined by Western blotanalysis. Culture media (0.5 ml) was methanol/chloroform precipitated,and the pellet was resuspended in SDS-PAGE sample buffer. Samples wereheated for 5 minutes at 95° C. before being applied to a 15% acrylamidegel. After SDS-PAGE, proteins were transferred to Protan nitrocellulosemembranes (Schleicher & Schuell, Germany) as described by Bass and Yang,in Creighton, T. E., ed., Protein Function: A Practical Approach, 2ndEdn., IRL Press, Oxford (1997), pp. 29-55. The membrane was blocked with1% bovine albumin (Sigma) in TBS (10×TBS per liter: 87.7 g NaCl, 66.1 gTrizma hydrochloride (Sigma) and 9.7 g Trizma base (Sigma), pH 7.4) for1 hour at room temperature, followed by an incubation with an anti-GP140or GP-160 antibody for 1 hour. The blot was washed 3 times for 10minutes with TBS-T (TBS with 0.05% Tween20), and incubated for 1 hourwith an alkaline-phosphatase-anti-mouse/rabbit/monkey/human IgGconjugate. After washing 2 times for 10 minutes with TBS-T and 2 timesfor 10 minutes with TBS, the development reaction was carried out usingalkaline phosphatase detection reagents (10 ml AP buffer (100 mMTris/HCl, 100 mM NaCl, pH 9.5) with 50 μl NBT solution (7.7% Nitro BlueTetrazolium (Sigma) in 70% dimethylformamide) and 37 μl of X-Phosphatesolution (5% of 5-bromo-4-chloro-3-indolyl phosphate indimethylformamide).

Example 15 Production and Purification of GP 140FOS

An anti-gp 120 antibody was covalently coupled to a NHS/EDC activateddextran and packed into a chromatography column. The supernatant,containing GP140FOS is loaded onto the column and after sufficientwashing, GP140FOS was eluted using 0.1 M HCl. The eluate was directlyneutralized during collection using 1 M Tris pH 7.2 in the collectiontubes.

Disulfide bond formation might occur during purification, therefore thecollected sample is treated with 10 mM DTT in 10 mM Tris pH 7.5 for 2hours at 25° C.

DTT is remove by subsequent dialysis against 10 mM Mes; 80 mM NaCl pH6.0. Finally GP140FOS is mixed with alphavirus particles containing theJUN leucine zipper in E2 as described in Example 16.

Example 16 Preparation of the AlphaVaccine Particles

Viral particles (see Examples 2 and 3) were concentrated using MilliporeUltrafree Centrifugal Filter Devices with a molecular weight cut-off of100 kD according to the protocol supplied by the manufacturer.Alternatively, viral particles were concentrated by sucrose gradientcentrifugation as described in the instruction manual of the SindbisExpression System (Invitrogen, San Diego, Calif.). The pH of the virussuspension was adjusted to 7.5 and viral particles were incubated in thepresence of 2-10 mM DTT for several hours. Viral particles were purifiedfrom contaminating protein on a Sephacryl S-300 column (Pharmacia)(viral particles elute with the void volume) in an appropriate buffer.

Purified virus particles were incubated with at least 240 fold molarexcess of FOS-antigen fusion protein in an appropriate buffer (pH7.5-8.5) in the presence of a redox shuffle (oxidizedglutathione/reduced glutathione; cystine/cysteine) for at least 10 hoursat 4° C. After concentration of the particles using a MilliporeUltrafree Centrifugal Filter Device with a molecular weight cut-off of100 kD, the mixture was passed through a Sephacryl S-300 gel filtrationcolumn (Pharmacia). Viral particles were eluted with the void volume.

Example 17 Fusion of JUN Amphipathic Helix to the Amino Terminus ofHBcAg(1-144)

The JUN helix was fused to the amino terminus of the HBcAg amino acidsequence 1 to 144 (JUN-HBcAg construct). For construction of theJUN-HBcAg DNA sequence, the sequences encoding the JUN helix andHBcAg(1-144) were amplified separately by PCR. The JUN sequence wasamplified from the pJuFo plasmid using primers EcoRI-JUN(s) andJUN-SacII(as). The EcoRI-JUN(s) primer introduced an EcoRI site followedby a start ATG codon. The JUN-SacII(as) primer introduced a linkerencoding the amino acid sequence GAAGS. The HBcAg (1-144) sequence wasamplified from the pEco63 plasmid (obtained from ATCC No. 31518) usingprimers JUN-HBcAg(s) and HBcAg(1-144)Hind(as). JUN-HBcAg(s) contained asequence corresponding to the 3′ end of the sequence encoding the JUNhelix followed by a sequence encoding the GAAGS linker and the 5′ end ofthe HBcAg sequence. HBcAg(1-144)Hind(as) introduces a stop codon and aHindIII site after codon 144 of the HBcAg gene. For the PCR reactions,100 pmol of each oligo and 50 ng of the template DNAs were used in the50 μl reaction mixtures with 2 units of Pwo polymerase, 0.1 mM dNTPs and2 mM MgSO₄. For both reactions, temperature cycling was carried out asfollows: 94° C. for 2 minutes; and 30 cycles of 94° C. (1 minute), 50°C. (1 minute), 72° C. (2 minutes).

Primer sequences: EcoRI-JUN(s):(5′-CCGGAATTCATGTGCGGTGGTCGGATCGCCCGG-3′); (SEQ ID NO: 61)JUN-SacII(as): (5′-GTCGCTACCCGCGGCTCCGCAACCAACGTGGTTCATGAC-3′); (SEQ IDNO: 62) JUN-HBcAg(s):(5′-GTTGGTTGCGGAGCCGCGGGTAGCGACATTGACCCTTATAAAGAATTTGG-3′); (SEQ ID NO:63) HBcAg(1-144)Hind(as):(5′-CGCGTCCCAAGCTTCTACGGAAGCGTTGATAGGATAGG-3′). (SEQ ID NO: 64)

Fusion of the two PCR fragments was performed by PCR using primersEcoRI-JUN(s) and HBcAg(1-144)Hind(as). 100 pmol of each oligo was usedwith 100 ng of the purified PCR fragments in a 50 μl reaction mixturecontaining 2 units of Pwo polymerase, 0.1 mM dNTPs and 2 mM MgSO₄. PCRcycling conditions were: 94° C. for 2 minutes; and 35 cycles of 94° C.(1 minute), 50° C. (1 minute), 72° C. (2 minutes). The final PCR productwas analyzed by agarose gel electrophoresis, purified and digested for16 hours in an appropriate buffer with EcoRI and HindIII restrictionenzymes. The digested DNA fragment was ligated intoEcoRI/HindIII-digested pKK vector to generate pKK-JUN-HBcAg expressionvector. Insertion of the PCR product was analyzed by EcoRI/HindIIIrestriction analysis and by DNA sequencing of the insert.

Example 18 Fusion of JUN Amphipathic Helix to the Carboxy Terminus ofHBcAg(1-144)

The JUN helix was fused to the carboxy terminus of the HBcAg amino acidsequence 1 to 144 (HBcAg-JUN construct). For construction of theHBcAg-JUN DNA sequence, the sequences encoding the JUN helix andHBcAg(1-144) were amplified separately by PCR. The JUN sequence wasamplified from the pJuFo plasmid with primers SacII-JUN(s) andJUN-HindIII(as). SacII-JUN(s) introduced a linker encoding amino acidsLAAG. This sequence also contains a SacII site. JUN-HindIII(as)introduced a stop codon (TAA) followed by a HindIII site. TheHBcAg(1-144) DNA sequence was amplified from the pEco63 plasmid usingprimers EcoRI-HBcAg(s) and HBcAg(1-144)-JUN(as). EcoRI-HBcAg(s)introduced an EcoRI site prior to the Start ATG of the HBcAg codingsequence. HBcAg(1-144)-JUN(as) introduces a sequence encoding thepeptide linker (LAAG), which also contains a SacII site. For the PCRreactions, 100 pmol of each oligo and 50 ng of the template DNAs wereused in the 50 μl reaction mixtures with 2 units of Pwo polymerase, 0.1mM dNTPs and 2 mM MgSO₄. Temperature cycling was carried out as follows:94° C. for 2 minutes; and 30 cycles of 94° C. (1 minute), 50° C. (1minute), 72° C. (2 minutes).

Primer Sequences SacII-JUN(s): (SEQ ID NO: 65)(5′-CTAGCCGCGGGTTGCGGTGGTCGGATCGCCCGG-3′); JUN-HindIII(as): (SEQ ID NO:66) (5′-CGCGTCCCAAGCTTTTAGCAACCAACGTGGTTCATGAC-3′); EcoRI-HBcAg(s): (SEQID NO: 67) (5′-CCGGAATTCATGGACATTGACCCTTATAAAG-3′); and HBcAg-JUN(as):(SEQ ID NO: 68) (5′- CCGACCACCGCAACCCGCGGCTAGCGGAAGCGTTGATAGGATAGG-3′).

Fusion of the two PCR fragments was performed by PCR using primersEcoRI-HBcAg(s) and JUN-HindIII(as). For the PCR fusion, 100 pmol of eacholigo was used with 100 ng of the purified PCR fragments in a 50 μlreaction mixture containing 2 units of Pwo polymerase, 0.1 mM dNTPs and2 mM MgSO₄. PCR cycling conditions were: 94° C. for 2 minutes; and 35cycles of 94° C. (1 minute), 50° C. (1 minute), 72° C. (2 minutes). Thefinal PCR product was analyzed by agarose gel electrophoresis, anddigested for 16 hours in an appropriate buffer with EcoRI and HindIIIrestriction enzymes. The DNA fragment was gel purified and ligated intoEcoRI/HindIII-digested pKK vector to generate pKK-HBcAg-JUN expressionvector. Insertion of the PCR product was analyzed by EcoRI/HindIIIrestriction analysis and by DNA sequencing of the insert.

Example 19 Insertion of JUN Amphipathic Helix into the c/e1 Epitope ofHBcAg(1-144)

The c/e1 epitope (residues 72 to 88) of HBcAg is known to be located inthe tip region on the surface of the Hepatitis B virus capsid. A part ofthis region (residues 76 to 82) of the protein was genetically replacedby the JUN helix to provide an attachment site for antigens(HBcAg-JUNIns construct). The HBcAg-JUNIns DNA sequence was generated byPCRs: The JUN helix sequence and two sequences encoding HBcAg fragments(amino acid residues 1 to 75 and 83 to 144) were amplified separately byPCR. The JUN sequence was amplified from the pJuFo plasmid with primersBamHII-JUN(s) and JUN-SacII(as). BamHI-JUN(s) introduced a linkersequence encoding the peptide sequence GSGGG that also contains a BamHIsite. JUN-SacII(as) introduced a sequence encoding the peptide linkerGAAGS followed by a sequence complementary to the 3′ end of the JUNcoding sequence. The HBcAg(1-75) DNA sequence was amplified from thepEco63 plasmid using primers EcoRIHBcAg(s) and HBcAg75-JUN(as).EcoRIHBcAg(s) introduced an EcoRI site followed by a sequencecorresponding to the 5′ end of the HBcAg sequence. HBcAg75-JUN(as)introduced a linker encoding the peptide GSGGG after amino acid 75 ofHBcAg followed by a sequence complementary to the 5′ end of the sequenceencoding the JUN helix. The HBcAg (83-144) fragment was amplified usingprimers JUN-HBcAg83(s) and HBcAg(1-144)Hind(as). JUN-HBcAg83(s)contained a sequence corresponding to the 3′ end of the JUN-encodingsequence followed by a linker encoding the peptide, GAAGS and a sequencecorresponding to the 5′ end of the sequence encoding HBcAg (83-144).HBcAg(1-144)Hind(as) introduced a stop codon and a HindIII site aftercodon 144 of the HBcAg gene. For the PCR reactions, 100 pmol of eacholigo and 50 ng of the template DNAs were used in the 50 μl reactionmixtures (2 units of Pwo polymerase, 0.1 mM dNTPs and 2 MM MgSO₄).Temperature cycling was performed as follows: 94° C. for 2 minutes; and35 cycles of 94° C. (1 minute), 50° C. (1 minute), 72° C. (2 minutes).

Primer sequences: BamHI-JUN(s): (SEQ ID NO: 69) (5′-CTAATGGATCCGGTGGGGGCTGCGGTGGTCGGATCGCCCGGCTCGAG- 3′); JUN-SacII(as):(SEQ ID NO: 70) (5′-GTCGCTACCCGCGGCTCCGCAACCAACGTGGTTCATGAC-3′);EcoRIHBcAg(s): (SEQ ID NO: 71) (5′-CCGGAATTCATGGACATTGACCCTTATAAAG-3′);HBcAg75-JUN (as): (SEQ ID NO: 72) (5′-CCGACCACCGCAGCCCCCACCGGATCCATTAGTACCCACCCAGGTAGC- 3′); JUN-HBcAg83(s):(SEQ ID NO: 73) (5′- GTTGGTTGCGGAGCCGCGGGTAGCGACCTAGTAGTCAGTTATGTC-3′);and HBcAg(1-144)Hind(as): (SEQ ID NO: 74)(5′-CGCGTCCCAAGCTTCTACGGAAGCGTTGATAGGATAGG-3′).

Fusion of the three PCR fragments was performed as follows. First, thefragment encoding HBcAg 1-75 was fused with the sequence encoding JUN byPCR using primers EcoRIHBcAg(s) and JUN-SacII(as). Second, the productobtained was fused with the HBcAg(83-144) fragment by PCR using primersEcoRI HBcAg(s) and HBcAg HindIII(as). For PCR fusions, 100 pmol of eacholigo was used with 100 ng of the purified PCR fragments in a 50 μlreaction mixture containing 2 units of Pwo polymerase, 0.1 mM dNTPs and2 mM MgSO₄. The same PCR cycles were used as for generation of theindividual fragments. The final PCR product was digested for 16 hours inan appropriate buffer with EcoRI and HindIII restriction enzymes. TheDNA fragment was ligated into EcoRI/HindIII-digested pKK vector,yielding the pKK-HBcAg-JUNIns vector. Insertion of the PCR product wasanalyzed by EcoRI/HindIII restriction analysis and by DNA sequencing ofthe insert.

Example 20 Fusion of the JUN Amphipathic Helix to the Carboxy Terminusof the Measles Virus Nucleocapsid (N) Protein

The JUN helix was fused to the carboxy terminus of the truncated measlesvirus N protein fragment comprising amino acid residues 1 to 473(N473-JUN construct). For construction of the DNA sequence encodingN473-JUN the sequence encoding the JUN helix and the sequence encodingN473-JUN were amplified separately by PCR. The JUN sequence wasamplified from the pJuFo plasmid with primers SacII-JUN(s) andJUN-HindIII(as). SacII-JUN(s) introduced a sequence encoding peptidelinker LAAG. This sequence also contained a SacII site. TheJUN-HindIII(as) anti-sense primer introduced a stop codon (TAA) followedby a HindIII site. The N (1-473) sequence was amplified from the pSC-Nplasmid containing the complete measles virus N protein coding sequence(obtained from M. Billeter, Zurich) using primers EcoRI-Nmea(s) andNmea-JUN(as). EcoRI-N(mea)(s) introduced an EcoRI site prior to theStart ATG of the N coding sequence. N(mea)-JUN(as) was complementary tothe 3′ end of the N(1-473) coding sequence followed by a sequencecomplementary to the coding sequence for the peptide linker (LAAG). Forthe PCR reactions, 100 pmol of each oligo and 50 ng of the template DNAswere used in the 50 μl reaction mixtures with 2 units of Pwo polymerase,0.1 mM dNTPs and 2 mM MgSO₄. Temperature cycling was performed asfollows: 94° C. for 2 minutes; and 35 cycles of 94° C. (1 minute), 55°C. (1 minute), 72° C. (2 minutes).

Primer sequences: SacII-JUN(s): (SEQ ID NO: 75)(5′-CTAGCCGCGGGTTGCGGTGGTCGGATCGCCCGG-3′); JUN-HindIII(as): (SEQ ID NO:76) (5′-CGCGTCCCAAGCTTTTAGCAACCAACGTGGTTCATGAC-3′); EcoRI-Nmea(s): (SEQID NO: 77) (5′-CCGGAATTCATGGCCACACTTTTAAGGAGC-3′); and Nmea-JUN(as):(SEQ ID NO: 78) (5′-CGCGTCCCAAGCTTTTAGCAACCAACGTGGTTCATGAC-3′).

Fusion of the two PCR fragments was performed in a further PCR usingprimers EcoRI-Nmea(s) and Nmea-JUN(as). For the PCR fusion, 100 pmol ofeach oligo was used with 100 ng of the purified PCR fragments in a 50 μlreaction mixture containing 2 units of Pwo polymerase, 0.1 mM dNTPs and2 mM MgSO₄. Temperature cycling was performed as follows: 94° C. for 2minutes; and 35 cycles of 94° C. (1 minute), 50° C. (1 minute), 72° C.(2 minutes). The PCR product was digested for 16 hours in an appropriatebuffer with EcoRI and HindIII restriction enzymes. The DNA fragment wasgel purified and ligated into EcoRI/HindIII-digested pKK vector,yielding the pKK-N473-JUN plasmid. Insertion of the PCR product wasanalyzed by EcoRI/HindIII restriction analysis and by DNA sequencing ofthe insert.

Example 21 Expression and Partial Purification of HBcAg-JUN

E. coli strain XL-1 blue was transformed with pKK-HBcAg-JUN. 1 ml of anovernight culture of bacteria was used to innoculate 100 ml of LB mediumcontaining 100 μ/ml ampicillin. This culture was grown for 4 hours at37° C. until an OD at 600 nm of approximately 0.8 was reached. Inductionof the synthesis of HBcAg-JUN was performed by addition of IPTG to afinal concentration of 1 mM. After induction, bacteria were furthershaken at 37° C. for 16 hours. Bacteria were harvested by centrifugationat 5000×g for 15 minutes. The pellet was frozen at −20° C. The pelletwas thawed and resuspended in bacteria lysis buffer (10 mM Na₂HPO₄, pH7.0, 30 mM NaCl, 0.25% Tween-20, 10 mM EDTA, 10 mM DTT) supplementedwith 200 μg/ml lysozyme and 10 μl of Benzonase (Merck). Cells wereincubated for 30 minutes at room temperature and disrupted using aFrench pressure cell. Triton X-100 was added to the lysate to a finalconcentration of 0.2%, and the lysate was incubated for 30 minutes onice and shaken occasionally. FIG. 4 shows HBcAg-JUN protein expressionin E. coli upon induction with IPTG. E. coli cells harboringpKK-HBcAg-JUN expression plasmid or a control plasmid were used forinduction of HBcAg-JUN expression with IPTG. Prior to the addition ofIPTG, a sample was removed from the bacteria culture carrying thepKK-HBcAg-JUN plasmid (lane 3) and from a culture carrying the controlplasmid (lane 1). Sixteen hours after addition of IPTG, samples wereagain removed from the culture containing pKK-HBcAg-JUN (lane 4) andfrom the control culture (lane 2). Protein expression was monitored bySDS-PAGE followed by Coomassie staining.

The lysate was then centrifuged for 30 minutes at 12,000×g in order toremove insoluble cell debris. The supernatant and the pellet wereanalyzed by Western blotting using a monoclonal antibody against HBcAg(YVS 1841, purchased from Accurate Chemical and Scientific Corp.,Westbury, N.Y., USA), indicating that a significant amount of HBcAg-JUNprotein was soluble (FIG. 5). Briefly, lysates from E. coli cellsexpressing HBcAg-JUN and from control cells were centrifuged at 14,000×gfor 30 minutes. Supernatant (=soluble fraction) and pellet (=insolublefraction) were separated and diluted with SDS sample buffer to equalvolumes. Samples were analyzed by SDS-PAGE followed by Western blottingwith anti-HBcAg monoclonal antibody YVS 1841. Lane 1: soluble fraction,control cells; lane 2: insoluble fraction, control cells; lane 3:soluble fraction, cells expressing HBcAg-JUN; lane 4: insolublefraction, cells expressing HbcAg-JUN.

The cleared cell lysate was used for step-gradient centrifugation usinga sucrose step gradient consisting of a 4 ml 65% sucrose solutionoverlaid with 3 ml 15% sucrose solution followed by 4 ml of bacteriallysate. The sample was centrifuged for 3 hrs with 100,000×g at 4° C.After centrifugation, 1 ml fractions from the top of the gradient werecollected and analyzed by SDS-PAGE followed by Coomassie staining. (FIG.6). Lane 1: total E. coli lysate prior to centrifugation. Lane 1 and 2:fractions 1 and 2 from the top of the gradient. Lane 4 to 7: fractions 5to 8 (15% sucrose). The HBcAg-JUN protein was detected by Coomassiestaining.

The HBcAg-JUN protein was enriched at the interface between 15 and 65%sucrose indicating that it had formed a capsid particle. Most of thebacterial proteins remained in the sucrose-free upper layer of thegradient, therefore step-gradient centrifugation of the HBcAg-JUNparticles led both to enrichment and to a partial purification of theparticles.

Example 22 Covalent Coupling of hGH-FOS to HBcAg-JUN

In order to demonstrate binding of a protein to HBcAg-JUN particles, wechose human growth hormone (hGH) fused with its carboxy terminus to theFOS helix as a model protein (hGH-FOS). HBcAg-JUN particles were mixedwith partially purified hGH-FOS and incubated for 4 hours at 4° C. toallow binding of the proteins. The mixture was then dialyzed overnightagainst a 3000-fold volume of dialysis buffer (150 mM NaCl, 10 mMTris-HCl solution, pH 8.0) in order to remove DTT present in both theHBcAg-JUN solution and the hGH-FOS solution and thereby allow covalentcoupling of the proteins through the establishment of disulfide bonds.As controls, the HBcAg-JUN and the hGH-FOS solutions were also dialyzedagainst dialysis buffer. Samples from all three dialyzed proteinsolutions were analyzed by SDS-PAGE under non-reducing conditions.Coupling of hGH-FOS to HBcAg-JUN was detected in an anti-hGH immunoblot(FIG. 7). hGH-FOS bound to HBcAg-JUN should migrate with an apparentmolecular mass of approximately 53 kDa, while unbound hGH-FOS migrateswith an apparent molecular mass of 31 kDa. The dialysate was analyzed bySDS-PAGE in the absence of reducing agent (lane 3) and in the presenceof reducing agent (lane 2) and detected by Coomassie staining. As acontrol, hGH-FOS that had not been mixed with capsid particles was alsoloaded on the gel in the presence of reducing agent (lane 1).

A shift of hGH-FOS to a molecular mass of approximately 53 kDa wasobserved in the presence of HBcAg-JUN capsid protein, suggesting thatefficient binding of hGH-FOS to HBcAg-JUN had taken place.

Example 23 Insertion of a Peptide Containing a Lysine Residue Into thec/e1 Epitope of HBcAg(1-149)

The c/e1 epitope (residues 72 to 88) of HBcAg is located in the tipregion on the surface of the Hepatitis B virus capsid (HBcAg). A part ofthis region (Proline 79 and Alanine 80) was genetically replaced by thepeptide Gly-Gly-Lys-Gly-Gly (HBcAg-Lys construct). The introduced Lysineresidue contains a reactive amino group in its side chain that can beused for intermolecular chemical crosslinking of HBcAg particles withany antigen containing a free cysteine group.

HBcAg-Lys DNA, having the amino acid sequence shown in SEQ ID NO:158,was generated by PCRs: The two fragments encoding HBcAg fragments (aminoacid residues 1 to 78 and 81 to 149) were amplified separately by PCR.The primers used for these PCRs also introduced a DNA sequence encodingthe Gly-Gly-Lys-Gly-Gly peptide. The HBcAg (1 to 78) fragment wasamplified from pEco63 using primers EcoRIHBcAg(s) and Lys-HBcAg(as). TheHBcAg (81 to 149) fragment was amplified from pEco63 using primersLys-HBcAg(s) and HBcAg(1-149)Hind(as). Primers Lys-HBcAg(as) andLys-HBcAg(s) introduced complementary DNA sequences at the ends of thetwo PCR products allowing fusion of the two PCR products in a subsequentassembly PCR. The assembled fragments were amplified by PCR usingprimers EcoRIHBcAg(s) and HbcAg(1-149)Hind(as).

For the PCRS, 100 pmol of each oligo and 50 ng of the template DNAs wereused in the 50 μl reaction mixtures with 2 units of Pwo polymerase, 0.1mM dNTPs and 2 mM MgSO4. For both reactions, temperature cycling wascarried out as follows: 94° C. for 2 minutes; 30 cycles of 94° C. (1minute), 50° C. (1 minute), 72° C. (2 minutes).

Primer sequences: EcoRIHBcAg(s): (SEQ ID NO: 79)(5′-CCGGAATTCATGGACATTGACCCTTATAAAG-3′); Lys-HBcAg(as): (SEQ ID NO: 80)(5′-CCTAGAGCCACCTTTGCCACCATCTTCTAAATTAGTACCCACCCAG GTAGC-3′);Lys-HBcAg(s): (SEQ ID NO: 81)(5′-GAAGATGGTGGCAAAGGTGGCTCTAGGGACCTAGTAGTCAGTTAT GTC-3′);HBcAg(1-149)Hind(as): (SEQ ID NO: 82)(5′-CGCGTCCCAAGCTTCTAAACAACAGTAGTCTCCGGAAG-3′).

For fusion of the two PCR fragments by PCR 100 pmol of primersEcoRIHBcAg(s) and HBcAg(1-149)Hind(as) were used with 100 ng of the twopurified PCR fragments in a 50 μl reaction mixture containing 2 units ofPwo polymerase, 0.1 mM dNTPs and 2 mM MgSO₄. PCR cycling conditionswere: 94° C. for 2 minutes; 30 cycles of 94° C. (1 minute), 50° C. (1minute), 72° C. (2 minutes). The assembled PCR product was analyzed byagarose gel electrophoresis, purified and digested for 19 hours in anappropriate buffer with EcoRI and HindIII restriction enzymes. Thedigested DNA fragment was ligated into EcoRI/HindIII-digested pKK vectorto generate pKK-HBcAg-Lys expression vector. Insertion of the PCRproduct into the vector was analyzed by EcoRI/HindIII restrictionanalysis and DNA sequencing of the insert.

The amino acid sequence of the HBcAg-Lys polypeptide isMDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREAIESPEHCSPHHTALRQAILCWGELMTLATWVGTNLEDGGKGGSRDLVVSYVNTNM GLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTL PETTVV (SEQ ID NO: 185). Thissequence differs from SEQ ID NO:134 at amino acid 74 (N in SEQ IDNO:1314, T in SEQ ID NO:185) and at amino acid 87 (N in SEQ ID NO:134, Sin SEQ ID NO: 185).

Example 24 Expression and Partial Purification of HBcAg-Lys

E. coli strain XL-1 blue was transformed with pKK-HBcAg-Lys. 1 ml of anovernight culture of bacteria was used to innoculate 100 ml of LB mediumcontaining 100 μg/ml ampicillin. This culture was grown for 4 hours at37° C. until an OD at 600 nm of approximately 0.8 was reached. Inductionof the synthesis of HBcAg-Lys was performed by addition of IPTG to afinal concentration of 1 mM. After induction, bacteria were furthershaken at 37° C. for 16 hours. Bacteria were harvested by centrifugationat 5000×g for 15 minutes. The pellet was frozen at −20° C. The pelletwas thawed and resuspended in bacteria lysis buffer (10 mM Na₂HPO₄, pH7.0, 30 mM NaCl, 0.25% Tween-20, 10 mM EDTA, 10 mM DTT) supplementedwith 200 μg/ml lysozyme and 10 μl of Benzonase (Merck). Cells wereincubated for 30 minutes at room temperature and disrupted using aFrench pressure cell. Triton X-100 was added to the lysate to a finalconcentration of 0.2%, and the lysate was incubated for 30 minutes onice and shaken occasionally. E. coli cells harboring pKK-HBcAg-Lysexpression plasmid or a control plasmid were used for induction ofHBcAg-Lys expression with IPTG. Prior to the addition of IPTG, a samplewas removed from the bacteria culture carrying the pKK-HBcAg-Lys plasmidand from a culture carrying the control plasmid. Sixteen hours afteraddition of IPTG, samples were again removed from the culture containingpKK-HBcAg-Lys and from the control culture. Protein expression wasmonitored by SD S-PAGE followed by Coomassie staining.

The lysate was then centrifuged for 30 minutes at 12,000×g in order toremove insoluble cell debris. The supernatant and the pellet wereanalyzed by Western blotting using a monoclonal antibody against HBcAg(YVS1841, purchased from Accurate Chemical and Scientific Corp.,Westbury, N.Y., USA), indicating that a significant amount of HBcAg-Lysprotein was soluble. Briefly, lysates from E. coli cells expressingHBcAg-Lys and from control cells were centrifuged at 14,000×g for 30minutes. Supernatant (=soluble fraction) and pellet (=insolublefraction) were separated and diluted with SDS sample buffer to equalvolumes. Samples were analyzed by SDS-PAGE followed by Western blottingwith anti-HBcAg monoclonal antibody YVS 1841.

The cleared cell lysate was used for step-gradient centrifugation usinga sucrose step gradient consisting of a 4 ml 65% sucrose solutionoverlaid with 3 ml 15% sucrose solution followed by 4 ml of bacteriallysate. The sample was centrifuged for 3 hrs with 100,000×g at 4° C.After centrifugation, 1 ml fractions from the top of the gradient werecollected and analyzed by SDS-PAGE followed by Coomassie staining. TheHBcAg-Lys protein was detected by Coomassie staining.

The HBcAg-Lys protein was enriched at the interface between 15 and 65%sucrose indicating that it had formed a capsid particle. Most of thebacterial proteins remained in the sucrose-free upper layer of thegradient, therefore step-gradient centrifugation of the HBcAg-Lysparticles led both to enrichment and to a partial purification of theparticles.

Example 25 Chemical Coupling of FLAG Peptide to HBcAg-Lys Using theHeterobifunctional Cross-Linker SPDP

Synthetic FLAG peptide with a Cysteine residue at its amino terminus(amino acid sequence CGGDYKDDDDK (SEQ ID NO:147)) was coupled chemicallyto purified HBcAg-Lys particles in order to elicit an immune responseagainst the FLAG peptide. 600 μl of a 95% pure solution of HBcAg-Lysparticles (2 mg/ml) were incubated for 30 minutes at room temperaturewith the heterobifunctional cross-linker N-Succinimidyl3-(2-pyridyldithio) propionate (SPDP) (0.5 mM). After completion of thereaction, the mixture was dialyzed overnight against 1 liter of 50 mMPhosphate buffer (pH 7.2) with 150 mM NaCl to remove free SPDP. Then 500μl of derivatized HBcAg-Lys capsid (2 mg/ml) were mixed with 0.1 mM FLAGpeptide (containing an amino-terminal cysteine) in the presence of 10 mMEDTA to prevent metal-catalyzed sulfhydryl oxidation. The reaction wasmonitored through the increase of the optical density of the solution at343 nm due to the release of pyridine-2-thione from SPDP upon reactionwith the free cysteine of the peptide. The reaction of derivatized Lysresidues with the peptide was complete after approximately 30 minutes.

The FLAG decorated particles were injected into mice.

Example 26 Construction of pMPSV-gp140cys

The gp140 gene was amplified by PCR from pCytTSgp140FOS using oligosgp140CysEcoRI and SalIgp140. For the PCRs, 100 pmol of each oligo and 50ng of the template DNAs were used in the 50 μl reaction mixtures with 2units of Pwo polymerase, 0.1 mM dNTPs and 2 mM MgSO4. For bothreactions, temperature cycling was carried out as follows: 94° C. for 2minutes; 30 cycles of 94° C. (0.5 minutes), 55° C. (0.5 minutes), 72° C.(2 minutes).

The PCR product was purified using QiaEXII kit, digested with SalI/EcoRIand ligated into vector pMPSVHE cleaved with the same enzymes.

Oligo sequences: Gp140CysEcoRI: (SEQ ID NO: 83)5′-GCCGAATTCCTAGCAGCTAGCACCGAATTTATCTAA-3′; SalIgp140: (SEQ ID NO: 84)5′-GGTTAAGTCGACATGAGAGTGAAGGAGAAATAT-3′.

Example 27 Expression of pMPSVgp140Cys

pMPSVgp140Cys (20 μg) was linearized by restriction digestion. Thereaction was stopped by phenol/chloroform extraction, followed by anisopropanol precipitation of the linearized DNA. The restrictiondigestion was evaluated by agarose gel eletrophoresis. For thetransfection, 5.4 μg of linearized pMPSVgp140-Cys was mixed with 0.6 μgof linearized pSV2Neo in 30 μl H₂O and 30 μl of 1 M CaCl₂ solution wasadded. After addition of 60 μl phosphate buffer (50 mM HEPES, 280 mMNaCl, 1.5 mM Na₂ HPO₄, pH 7.05), the solution was vortexed for 5seconds, followed by an incubation at room temperature for 25 seconds.The solution was immediately added to 2 ml HP-1 medium containing 2% FCS(2% FCS medium). The medium of an 80% confluent BHK21 cell culture(6-well plate) was then replaced by the DNA containing medium. After anincubation for 5 hours at 37° C. in a CO₂ incubator, the DNA containingmedium was removed and replaced by 2 ml of 15% glycerol in 2% FCSmedium. The glycerol containing medium was removed after a 30 secondincubation phase, and the cells were washed by rinsing with 5 ml of HP-1medium containing 10% FCS. Finally 2 ml of fresh HP-l medium containing10% FCS was added.

Stably transfected cells were selected and grown in selection medium(HP-1 medium supplemented with G418) at 37° C. in a CO₂ incubator. Whenthe mixed population was grown to confluency, the culture was split totwo dishes, followed by a 12 h growth period at 37° C. One dish of thecells was shifted to 30° C. to induce the expression of solubleGP140-FOS. The other dish was kept at 37° C.

The expression of soluble GP140-Cys was determined by Western blotanalysis. Culture media (0.5 ml) was methanol/chloroform precipitated,and the pellet was resuspended in SDS-PAGE sample buffer. Samples wereheated for 5 minutes at 95° C. before being applied to a 15% acrylamidegel. After SDS-PAGE, proteins were transferred to Protan nitrocellulosemembranes (Schleicher & Schuell, Germany) as described by Bass and Yang,in Creighton, T. E., ed., Protein Function: A Practical Approach, 2ndEdn., IRL Press, Oxford (1997), pp. 29-55. The membrane was blocked with1% bovine albumin (Sigma) in TBS (10×TBS per liter: 87.7 g NaCl, 66.1 gTrizma hydrochloride (Sigma) and 9.7 g Trizma base (Sigma), pH 7.4) for1 hour at room temperature, followed by an incubation with an anti-GP140or GP-160 antibody for 1 hour. The blot was washed 3 times for 10minutes with TBS-T (TBS with 0.05% Tweeri20), and incubated for 1 hourwith an alkaline-phosphatase-anti-mouse/rabbit/monkey/human IgGconjugate. After washing 2 times for 10 minutes with TBS-T and 2 timesfor 10 minutes with TBS, the development reaction was carried out usingalkaline phosphatase detection reagents (10 ml AP buffer (100 mMTris/HCl, 100 mM NaCl, pH 9.5) with 50 μl NBT solution (7.7% Nitro BlueTetrazolium (Sigma) in 70% dimethylformamide) and 37 μl of X-Phosphatesolution (5% of 5-bromo-4-chloro-3-indolyl phosphate indimethylformamide).

Example 28 Purification of gp140Cys

An anti-gp120 antibody was covalently coupled to a NHS/EDC activateddextran and packed into a chromatography column. The supernatant,containing GP140Cys is loaded onto the column and after sufficientwashing, GP140Cys was eluted using 0.1 M HCl. The eluate was directlyneutralized during collection using 1 M Tris pH 7.2 in the collectiontubes.

Disulfide bond formation might occur during purification, therefore thecollected sample is treated with 10 mM DTT in 10 mM Tris pH 7.5 for 2hours at 25° C.

DTT is remove by subsequent dialysis against 10 mM Mes; 80 mM NaCl pH6.0. Finally GP140Cys is mixed with alphavirus particles containing theJUN residue in E2 as described in Example 16.

Example 29 Construction of PLA2-Cys

The PLA2 gene was amplified by PCR from pAV3PLAfos using oligos EcoRIPLAand PLA-Cys-hind. For the PCRs, 100 pmol of each oligo and 50 ng of thetemplate DNAs were used in the 50 μl reaction mixtures with 2 units ofPwo polymerase, 0.1 mM dNTPs and 2 mM MgSO4. For both reactions,temperature cycling was carried out as follows: 94° C. for 2 minutes; 30cycles of 94° C. (0.5 minutes), 55° C. (0.5 minutes), 72° C. (2minutes).

The PCR product was purified using QiaEXII kit, digested withEcoRI/HinDIII and ligated into vector pAV3 cleaved with the sameenzymes.

Oligos EcoRIPLA: (SEQ ID NO: 85) 5′-TAACCGAATTCAGGAGGTAAAAAGATATGG-3′PLACys-hind: (SEQ ID NO: 86) 5′-GAAGTAAAGCTTTTAACCACCGCAACCACCAGAAG-3′.

Example 30 Expression and Purification of PLA-Cys

For cytoplasmic production of Cys tagged proteins, E. coli XL-1-Bluestrain was transformed with the vectors pAV3::PLA and pPLA-Cys. Theculture was incubated in rich medium in the presence of ampicillin at37° C. with shaking. At an optical density (550 nm) of, 1 mM IPTG wasadded and incubation was continued for another 5 hours. The cells wereharvested by centrifugation, resuspended in an appropriate buffer (e.g.,Tris-HCl, pH 7.2, 150 mM NaCl) containing DNase, RNase and lysozyme, anddisrupted by passage through a french pressure cell. Aftercentrifugation (Sorvall RC-5C, SS34 rotor, 15000 rpm, 10 min, 4° C.),the pellet was resuspended in 25 ml inclusion body wash buffer (20 mMtris-HCl, 23% sucrose, 0.5% Triton X-100, 1 mM EDTA, pH8) at 4° C. andrecentrifuged as described above. This procedure was repeated until thesupernatant after centrifugation was essentially clear. Inclusion bodieswere resuspended in 20 ml solubilization buffer (5.5 M guanidiniumhydrochloride, 25 mM tris-HCl, pH 7.5) at room temperature and insolublematerial was removed by centrifugation and subsequent passage of thesupernatant through a sterile filter (0.45 μm). The protein solution waskept at 4° C. for at least 10 hours in the presence of 10 mM EDTA and100 mM DTT and then dialyzed three times against 10 volumes of 5.5 Mguanidinium hydrochloride, 25 mM tris-HCl, 10 mM EDTA, pH 6. Thesolution was dialyzed twice against 51 2 M urea, 4 mM EDTA, 0.1 M NH₄Cl,20 mM sodium borate (pH 8.3) in the presence of an appropriate redoxshuffle (oxidized glutathione/reduced glutathione; cystine/cysteine).The refolded protein was then applied to an ion exchange chromatography.The protein was stored in an appropriate buffer with a pH above 7 in thepresence of 2-10 mM DTT to keep the cysteine residues in a reduced form.Prior to coupling of the protein with the alphavirus particles, DTT wasremoved by passage of the protein solution through a Sephadex G-25 gelfiltration column.

Example 31 Construction of a HBcAg Devoid of Free Cysteine Residues andContaining an Inserted Lysine Residue

A Hepatitis core Antigen (HBcAg), referred to herein asHBcAg-lys-2cys-Mut, devoid of cysteine residues at positionscorresponding to 48 and 107 in SEQ ID NO:134 and containing an insertedlysine residue was constructed using the following methods.

The two mutations were introduced by first separately amplifying threefragments of the HBcAg-Lys gene prepared as described above in Example23 with the following PCR primer combinations. PCR methods essentiallyas described in Example 1 and conventional cloning techniques were usedto prepare the HBcAg-lys-2cys-Mut gene.

In brief, the following primers were used to prepare fragment 1: Primer1: EcoRIHBcAg(s) (SEQ ID NO: 148) CCGGAATTCATGGACATTGACCCTTATAAAG Primer2: 48as (SEQ ID NO: 149) GTGCAGTATGGTGAGGTGAGGAATGCTCAGGAGACTC

The following primers were used to prepare fragment 2: Primer 3: 48s(SEQ ID NO: 150) GSGTCTCCTGAGCATTCCTCACCTCACCATACTGCAC Primer 4: 107as(SEQ ID NO: 151) CTTCCAAAAGTGAGGGAAGAAATGTGAAACCAC The following primerswere used to prepare fragment 3: Primer 5: HBcAg149hind-as (SEQ ID NO:152) CGCGTCCCAAGCTTCTAAACAACAGTAGTCTCCGGAAGCGTTGATAG Primer 6: 107s (SEQID NO: 153) GTGGTTTCACATTTCTTCCCTCACTTTTGGAAG

Fragments 1 and 2 were then combined with PCR primers EcoRIHBcAg(s) and107as to give fragment 4. Fragment 4 and fragment 3 were then combinedwith primers EcoRIHBcAg(s) and HBcAg149hind-as to produce the fulllength gene. The full length gene was then digested with the EcoRI(GAATTC) and HindIII (AAGCTT) enzymes and cloned into the pKK vector(Pharmacia) cut at the same restriction sites. The amino acid sequenceof the HBcAg-Lys-2cys-Mut polypeptide is MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHSSPHHTALRQAILCWGELMTLATWVGTNLEDGGKGGSRDLVVSYVNTNMGLKIRQLLWFHISSLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVV (SEQ ID NO:186).

Example 32 Blockage of Free Cysteine Residues of a HBcAg Followed byCross-Linking

The free cysteine residues of the HBcAg-Lys prepared as described abovein Example 23 were blocked using Iodacetamide. The blocked HBcAg-Lys wasthen cross-linked to the FLAG peptide with the hetero-bifunctionalcross-linker m-maleimidonbenzoyl-N-hydroxysuccinimide ester (Sulfo-MBS).

The methods used to block the free cysteine residues and cross-link theHBcAg-Lys are as follows. HBcAg-Lys (550 μg/ml) was reacted for 15minutes at room temperature with Iodacetamide (Fluka Chemie, Brugg,Switzerland) at a concentration of 50 mM in phosphate buffered saline(PBS) (50 mM sodium phosphate, 150 mM sodium chloride), pH 7.2, in atotal volume of 1 ml. The so modified HBcAg-Lys was then reactedimmediately with Sulfo-MBS (Pierce) at a concentration of 530 μMdirectly in the reaction mixture of step 1 for 1 hour at roomtemperature. The reaction mixture was then cooled on ice, and dialyzedagainst 1000 volumes of PBS pH 7.2. The dialyzed reaction mixture wasfinally reacted with 300 μM of the FLAG peptide (CGGDYKDDDDK (SEQ IDNO:147)) containing an N-terminal free cysteine for coupling to theactivated HBcAg-Lys, and loaded on SDS-PAGE for analysis.

As shown in FIG. 8, the resulting patterns of bands on the SDS-PAGE gelshowed a clear additional band migrating slower than the controlHBcAg-Lys derivatized with the cross-linker, but not reacted with theFLAG peptide. Reactions done under the same conditions without priorderivatization of the cysteines with lodacetamide led to completecross-linking of monomers of the HBcAg-Lys to higher molecular weightspecies.

Example 33 Isolation of Type-1 Pili and Chemical Coupling of FLAGPeptide to Type-1 Pili of Escherichia coli using a HeterobifunctionalCross-Linker

A. Introduction

Bacterial pili or fimbriae are filamentous surface organelles producedby a wide range of bacteria. These organelles mediate the attachment ofbacteria to surface receptors of host cells and are required for theestablishment of many bacterial infections like cystitis,pyelonephritis, new born meningitis and diarrhea.

Pili can be divided in different classes with respect to their receptorspecificity (agglutination of blood cells from different species), theirassembly pathway (extracellular nucleation, general secretion,chaperone/usher, alternate chaperone) and their morphological properties(thick, rigid pili; thin, flexible pili; atypical structures includingcapsule; curli; etc). Examples of thick, rigid pili forming a righthanded helix that are assembled via the so called chaperone/usherpathway and mediate adhesion to host glycoproteins include Type-1 pili,P-pili, S-pili, FIC-pili, and 987P-pili). The most prominent and bestcharacterized members of this class of pili are P-pili and Type-1 pili(for reviews on adhesive structures, their assembly and the associateddiseases see Soto, G. E. & Hultgren, S. J., J. Bacteriol. 181: 1059-1071(1999); Bullitt & Makowski, Biophys. J. 74: 623-632 (1998); Hung, D. L.& Hultgren, S. J., J. Struct, Biol. 124: 201-220 (1998)).

Type-1 pili are long, filamentous polymeric protein structures on thesurface of E. coli. They possess adhesive properties that allow forbinding to mannose-containing receptors present on the surface ofcertain host tissues. Type-1 pili can be expressed by 70-80% of all E.coli isolates and a single E. coli cell can bear up to 500 pili.Type-pili reach a length of typically 0.2 to 2 μM with an average numberof 1000 protein subunits that associate to a right-handed helix with3.125 subunits per turn with a diameter of 6 to 7 nm and a central holeof 2.0 to 2.5 nm.

The main Type-1 pilus component, FimA, which represents 98% of the totalpilus protein, is a 15.8 kDa protein. The minor pilus components FimF,FimG and FimH are incorporated at the tip and in regular distances alongthe pilus shaft (Klemm, P. & Krogfelt, K. A., “Type 1 fimbriae ofEscherichia coli,” in: Fimbriae. Klemm, P. (ed.), CRC Press Inc., (1994)pp. 9-26). FimH, a 29.1 kDa protein, was shown to be the mannose-bindingadhesin of Type-1 pili (Krogfelt, K. A., et al., Infect. Immun. 58:1995-1998 (1990); Klemm, P., et al., Mol. Microbiol. 4: 553-560 (1990);Hanson, M. S. & Brinton, C. C. J., Nature 17: 265-268 (1988)), and itsincorporation is probably facilitated by FimG and FimF (Klemm, P. &Christiansen, G., Mol. Gen. Genetics 208: 439-445 (1987); Russell, P. W.& Orndorff, P. E., J. Bacteriol. 174: 5923-5935 (1992)). Recently, itwas shown that FimH might also form a thin tip-fibrillum at the end ofthe pili (Jones, C. H., et al., Proc. Nat. Acad. Sci. USA 92: 2081-2085(1995)). The order of major and minor components in the individualmature pili is very similar, indicating a highly ordered assemblyprocess (Soto, G. E. & Hultgren, S. J., J. Bacteriol 181: 1059-1071(1999)).

P-pili of E. coli are of very similar architecture, have a diameter of6.8 nm, an axial hole of 1.5 nm and 3.28 subunits per turn (Bullitt &Makowski, Biophys. J. 74: 623-632 (1998)). The 16.6 kDa PapA is the maincomponent of this pilus type and shows 36% sequence identity and 59%similarity to FimA (see Table 1). As in Type-1 pili the 36.0 kDa P-pilusadhesin PapG and specialized adapter proteins make up only a tinyfraction of total pilus protein. The most obvious difference to Type-1pili is the absence of the adhesin as an integral part of the pilus rod,and its exclusive localization in the tip fibrillium that is connectedto the pilus rod via specialized adapter proteins that Type-1 pili lack(Hultgren, S. J., et al., Cell 73: 887-901 (1993)). TABLE 1 Similarityand identity between several structural pilus proteins of Type-1 andP-pili (in percent). The adhesins were omitted. Similarity FimA PapAFimI FimF FimG PapE PapK PapH PapF Identity FimA 59 57 56 44 50 44 46 46PapA 36 49 48 41 45 49 49 47 FimI 35 31 56 46 40 47 48 48 FimF 34 26 3040 47 43 49 48 FimG 28 28 28 26 39 39 41 45 PapE 25 23 18 28 22 43 47 54PapK 24 29 25 28 22 18 49 53 PapH 22 26 22 22 23 24 23 41 PapF 18 22 2224 28 27 26 21

Type-1 pili are extraordinary stable hetero-oligomeric complexes.Neither SDS-treatment nor protease digestions, boiling or addition ofdenaturing agents can dissociate Type-1 pili into their individualprotein components. The combination of different methods like incubationat 100° C. at pH 1.8 was initially found to allow for thedepolymerization and separation of the components (Eshdat, Y., et al.,J. Bacteriol. 148: 308-314 (1981); Brinton, C. C. J., Trans, N. Y Acad.Sci. 27: 1003-1054 (1965); Hanson, A. S., et al, J. Bacteriol., 170:3350-3358 (1988); Klemm, P. & Krogfelt, K. A., “Type I fimbriae ofEscherichia coli,” in: Fimbriae. Klemm, P. (ed.), CRC Press Inc., (1994)pp. 9-26). Interestingly, Type-1 pili show a tendency to break atpositions where FimH is incorporated upon mechanical agitation,resulting in fragments that present a FimH adhesin at their tips. Thiswas interpreted as a mechanism of the bacterium to shorten pili to aneffective length under mechanical stress (Klemm, P. & Krogfelt, K. A.,“Type I fimbriae of Escherichia coli,” in: Fimbriae. Klemm, P. (ed.),CRC Press Inc., (1994) pp. 9-26). Despite their extraordinary stability,Type-1 pili have been shown to unravel partially in the presence of 50%glycerol; they lose their helical structure and form an extended andflexible, 2 nm wide protein chain (Abraham, S. N., et al., J. Bacteriol174: 5145-5148 (1992)).

P-pili and Type-1 pili are encoded by single gene clusters on the E.coli chromosome of approximately 10 kb (Klemm, P. & Krogfelt, K. A.,“Type I fimbriae of Escherichia coli,” in: Fimbriae. Klemm, P. (ed.),CRC Press Inc., (1994) pp. 9-26; Orndorff, P. E. & Falkow, S., J.Bacteriol. 160: 61-66 (1984)). A total of nine genes are found in theType-1 pilus gene cluster, and 11 genes in the P-pilus cluster(Hultgren, S. J., et al., Adv. Prot. Chem. 44: 99-123 (1993)). Bothclusters are organized quite similarly.

The first two fim-genes, fimB and fimE, code for recombinases involvedin the regulation of pilus expression (McClain, M. S., et al., J.Bacteriol. 173: 5308-5314(1991)). The main structural pilus protein isencoded by the next gene of the cluster, fimA (Klemm, P., Euro. J.Biochem. 143: 395-400 (1984); Orndorff, P. E. & Falkow, S., J.Bacteriol. 160: 61-66 (1984); Orndorff, P. E. & Falkow, S., J.Bacteriol. 162: 454-457 (1985)). The exact role of fim is unclear. Ithas been reported to be incorporated in the pilus as well (Klemm, P. &Krogfelt, K. A., “Type I fimbriae of Escherichia coli,” in: Fimbriae.Klemm, P. (ed.), CRC Press Inc., (1994) pp. 9-26). The adjacent fimCcodes not for a structural component of the mature pilus, but for aso-called pilus chaperone that is essential for the pilus assembly(Klemm, P., Res. Microbiol. 143: 831-838 (1992), Jones, C. H., et al.,Proc. Nat. Acad Sci. USA 90: 8397-8401 (1993)).

The assembly platform in the outer bacterial membrane to which themature pilus is anchored is encoded by fimD (Klemm, P. & Christiansen,G., Mol. Gen, Genetics 220: 334-338 (1990)). The three minor componentsof the Type-1 pili, FimF, FimG and FimH are encoded by the last threegenes of the cluster (Klemm, P. & Christiansen, G., Mol. Gen. Genetics208: 439-445 (1987)). Apart from fimB and fimE, all genes encodeprecursor proteins for secretion into the periplasm via the sec-pathway.

The similarities between different pili following the chaperone/usherpathway are not restricted to their morphological properties. Theirgenes are also arranged in a very similar manner. Generally the gene forthe main structural subunit is found directly downstream of theregulatory elements at the beginning of the gene cluster, followed by agene for an additional structural subunit (fimI in the case of Type-1pili and papH in the case of P-pili). PapH was shown and FimI issupposed to terminate pilus assembly (Hultgren, S. J., et al., Cell 73:887-901 (1993)). The two proteins that guide the process of pilusformation, namely the specialized pilus chaperone and the outer membraneassembly platform, are located adjacently downstream. At the end of theclusters a variable number of minor pilus components including theadhesins are encoded. The similarities in morphological structure,sequence (see Table 1), genetic organization and regulation indicate aclose evolutionary relationship and a similar assembly process for thesecell organelles.

Bacteria producing Type-1 pili show a so-called phase-variation. Eitherthe bacteria are fully piliated or bald. This is achieved by aninversion of a 314 bp genomic DNA fragment containing the fimA promoter,thereby inducing an “all on” or “all off” expression of the pilus genes(McClain, M. S., et al., J. Bacteriol. 173: 5308-5314 (1991)). Thecoupling of the expression of the other structural pilus genes to fimAexpression is achieved by a still unknown mechanism. However, a widerange of studies elucidated the mechanism that influences the switchingbetween the two phenotypes.

The first two genes of the Type-1 pilus cluster, fimB and fimE encoderecombinases that recognize 9 bp DNA segments of dyad symmetry thatflank the invertable fimA promoter. Whereas FimB switches pilation “on”,FimE turns the promoter in the “off” orientation. The up- ordown-regulation of either fimB or fimE expression therefore controls theposition of the so-called “fim-switch” (McClain, M. S., et al., J.Bacteriol. 173: 5308-5314 (1991); Blomfield, I. C., et al., J.Bacteriol. 173: 5298-5307 (1991)).

The two regulatory proteins fimB and fimE are transcribed from distinctpromoters and their transcription was shown to be influenced by a widerange of different factors including the integration host factor (IHF)(Blomfield, I. C., et al., Mol. Microbiol, 23: 705-717 (1997)) and theleucine-responsive regulatory protein (LRP) (Blomfield, I. C., et al.,J. Bacteriol. 175: 27-36 (1993); Gally, D. L., et al., J. Bacteriol.175: 6186-6193 (1993); Gally, D. L., et al., Microbiol. 21: 725-738(1996); Roesch, R. L. & Blomfield, I. C., Mol. Microbiol, 27: 751-761(1998)). Mutations in the former lock the bacteria either in “on” or“off” phase, whereas LRP mutants switch with a reduced frequency. Inaddition, an effect of leuX on pilus biogenesis has been shown. Thisgene is located in the vicinity of the fim-genes on the chromosome andcodes for the minor leucine tRNA species for the UUG codon. Whereas fimBcontains five UUG codons, fimE contains only two, and enhanced leuXtranscription might favor FimB over FimE expression (Burghoff, R. L., etal., Infect. Immun. 61: 1293-1300 (1993); Newman, J. V., et al., FEMSMicrobiol. Lett. 122: 281-287 (1994); Ritter, A., et al., Mol.Microbial, 25: 871-882 (1997)).

Furthermore, temperature, medium composition and other environmentalfactors were shown to influence the activity of FimB and FimE. Finally,a spontaneous, statistical switching of the fimA promoter has beenreported. The frequency of this spontaneous switching is approximately10⁻³ per generation (Eisenstein, B. I., Science 214: 337-339 (1981);Abraham, S. M., et al., Proc. Nat. Acad. Sci, USA 82: 5724-5727 (1985)),but is strongly influenced by the above mentioned factors.

The genes fimI and fimC are also transcribed from the fimA promoter, butdirectly downstream of fimA a DNA segment with a strong tendency to formsecondary structure was identified which probably represents a partialtranscription terminator (Klemm, P., Euro. J. Biochem. 143: 395-400(1984)); and is therefore supposed to severely reduce fimI and fimCtranscription. At the 3′ end of fimC an additional promoter controls thefimD transcription; at the 3′ end of fimD the last known fim promoter islocated that regulates the levels of FimF, FimG, and FimH. Thus, all ofthe minor Type-1 pili proteins are transcribed as a single mRNA (Klemm,P. & Krogfelt, K. A., “Type I fimbriae of Escherichia coli,” in:Fimbriae. Klemm, P. (ed.), CRC Press Inc., (1994) pp. 9-26). Thisensures a 1:1:1 stochiometry on mRNA-level, which is probably maintainedon the protein level.

In the case of P-pili additional regulatory mechanisms were found whenthe half-life of mRNA was determined for different P-pilus genes. ThemRNA for papA was extraordinarily long-lived, whereas the mRNA for papB,a regulatory pilus protein, was encoded by short-lived mRNA(Naureckiene, S. & Uhlin. B. E., Mol. Microbiol. 21: 55-68 (1996);Nilsson, P., et al., J. Bacterial. 178: 683-690 (1996)).

In the case of Type-1 pili, the gene for the Type-1 pilus chaperone FimCstarts with a GTG instead of an ATG codon, leading to a reducedtranslation efficiency. Finally, analysis of the fimH gene revealed atendency of the fimH mRNA to form a stem-loop, which might severelyhamper translation. In summary, bacterial pilus biogenesis is regulatedby a wide range of different mechanisms acting on all levels of proteinbiosynthesis.

Periplasmic pilus proteins are generally synthesized as precursors,containing a N-terminal signal-sequence that allows translocation acrossthe inner membrane via the Sec-apparatus. After translocation theprecursors are normally cleaved by signal-peptidase I. Structural Type-1pilus subunits normally contain disulfide bonds, their formation iscatalyzed by DsbA and possibly DsbC and DsbG gene products.

The Type-1 pilus chaperone FimC lacks cysteine residues. In contrast,the chaperone of P-pili, PapD, is the only member of the pilus chaperonefamily that contains a disulfide bond, and the dependence of P-pili onDsbA has been shown explicitly (Jacob-Dubuisson, F., et al, Proc. Nat.Acad. Sci. USA 91: 11552-11556 (1994)). PapD does not accumulate in theperiplasm of a ΔdsbA strain, indicating that the disturbance of theP-pilus assembly machinery is caused by the absence of the chaperone(Jacob-Dubuisson, F., et al., Proc. Nat. Acad. Sci. USA 91: 11552-11556(1994)). This is in accordance with the finding that Type-1 pili arestill assembled in a ΔdsbA strain, albeit to reduced level (Hultgren, S.J., et al., “Bacterial Adhesion and Their Assembly”, in: Escherichiacoli and Salmonella, Neidhardt, F. C. (ed.) ASM Press, (1996) pp.2730-2756).

Type-1 pili as well as P-pili are to 98% made of a single or mainstructural subunit termed FimA and PapA, respectively. Both proteinshave a size of ˜15.5 kDa. The additional minor components encoded in thepilus gene clusters are very similar (see Table 1). The similarities insequence and size of the subunits with the exception of the adhesinssuggest that all share an identical folding motif, and differ only withrespect to their affinity towards each other. Especially the N- andC-terminal regions of these proteins are well conserved and supposed toplay an important role in chaperone/subunit interactions as well as insubunit/subunit interactions within the pilus (Soto, G. E. & Hultgren,S. J., J. Bacteriol. 181: 1059-1071 (1999)). Interestingly, theconserved N-terminal segment can be found in the middle of the pilusadhesins, indicating a two-domain organization of the adhesins where theproposed C-terminal domain, starting with the conserved motif,corresponds to a structural pilus subunit whereas the N-terminal domainwas shown to be responsible for recognition of host cell receptors(Hultgren, S. J., et al., Proc. Nat. Acad. Sci. USA 86: 4357-4361(1989), Haslam, D. B., et al., Mol. Microbiol. 14: 399-409 (1994); Soto,G. E., et al., EMBO J. 17: 6155-6167 (1998)). The different subunitswere also shown to influence the morphological properties of the pili.The removal of several genes was reported to reduce the number of Type-1or P-pili or to increase their length, (fimH, papG, papK, fimF, fimG)(Russell, P. W. & Orndorff, P. E., J. Bacteriol. 174: 5923-5935 (1992);Jacob-Dubuisson, R., et al., EMBO J. 12: 837-847 (1993); Soto, G. E. &Hultgren, S. J., J. Bacteriol. 181: 1059-1071 (1999)); combination ofthe gene deletions amplified these effects or led to a total loss ofpilation (Jacob-Dubuisson, R., et al., EMBO J. 12: 837-847 (1993)).

In non-fimbrial adhesive cell organelles also assembled viachaperones/usher systems such as Myf fimbriae and CS3 pili, theconserved C-terminal region is different. This indirectly proves theimportance of these C-terminal subunit segments for quaternaryinteractions (Hultgren, S. J., et al., “Bacterial Adhesion and TheirAssembly”, in: Escherichia coli and Salmonella, Neidhardt, F. C. (ed.)ASM Press, (1996) pp. 2730-2756).

Gene deletion studies proved that removal of the pilus chaperones leadsto a total loss of piliation in P-pili and Type-1 pili (Lindberg, F., etal., J. Bacteriol. 171: 6052-6058 (1989); Klemm, P., Res. Microbiol.143: 831-838 (1992); Jones, C. H., et al., Proc. Nat. Acad Sci. USA 90:8397-8401 (1993)). Periplasmic extracts of a ΔfimC strain showed theaccumulation of the main subunit FimA, but no pili could be detected(Klemm, P., Res. Microbiol. 143: 831-838 (1992)). Attempts toover-express individual P-pilus subunits failed and only proteolyticallydegraded forms could be detected in the absence of PapD; in addition,the P-pilus adhesin was purified with the inner membrane fraction in theabsence of the chaperone (Lindberg, F., et al., J. Bacteriol. 171:6052-6058 (1989)). However, co-expression of the structural pilusproteins and their chaperone allowed the detection of chaperone/subunitcomplexes from the periplasm in the case of the FimC/FimH complex aswell as in the case of different Pap-proteins including the adhesin PapGand the main subunit PapA (Tewari, R., et al., J. Biol. Chem. 268:3009-3015 (1993); Lindberg, F., et al., J. Bacteriol. 171: 6052-6058(1989)). The affinity of chaperone/subunit complexes towards theirassembly platform has also been investigated in vitro and was found todiffer strongly (Dodson et al., Proc. Natl Acad. Sci. USA 90: 3670-3674(1993)). From these results the following functions were suggested forthe pilus chaperones:

They are assumed to recognize unfolded pilus subunits, prevent theiraggregation and to provide a “folding template” that guides theformation of a native structure.

The folded subunits, which after folding display surfaces that allowsubunit/subunit interactions, are then expected to be shielded frominteracting with other subunits, and to be kept in a monomeric,assembly-competent state.

Finally, the pilus chaperones are supposed to allow a triggered releaseof the subunits at the outer membrane assembly location, and, by doingso with different efficiency, influence the composition and order of themature pili (see also the separate section below).

After subunit release at the outer membrane, the chaperone is free foranother round of substrate binding, folding assistance, subunittransport through the periplasm and specific delivery to the assemblysite. Since the periplasm lacks energy sources, like ATP, the wholepilus assembly process must be thermodynamically driven(Jacob-Dubuisson, F., et al., Proc. Nat. Acad. Sci. USA 91: 11552-11556(1994)). The wide range of different functions attributed to the piluschaperones would implicate an extremely fine tuned cascade of steps.

Several findings, however, are not readily explained with the model ofpilus chaperone function outlined above. One example is the existence ofmultimeric chaperone/subunit complexes (Striker, R. T., et al., J. Biol.Chem. 269: 12233-12239 (1994)), where one chaperone binds subunit dimersor trimers. It is difficult to imagine a folding template that can be“double-booked”. The studies on the molecular details ofchaperone/subunit interaction (see below) partially supported thefunctions summarized above, but also raised new questions.

All 31 periplasmic chaperones identified by genetic studies or sequenceanalysis so far are proteins of approximately 25 kDa with conspicuouslyhigh pI values around 10. Ten of these chaperones assist the assembly ofrod-like pili, four are involved in the formation of thin pili, ten areimportant for the biogenesis of atypically thin structures (includingcapsule-like structures) and two adhesive structures have not beendetermined so far (Holmgren, A., et al., EMBO J. 11: 1617-1622 (1992),Bonci, A., et al., J. Mol. Evolution 44: 299-309 (1997); Smyth, C. J.,et al, FEMS Immun. Med Microbiol. 16: 127-139 (1996); Hung, D. L. &Hultgren, S. J., J. Struct, Biol. 124: 201-220 (1998)). The pairwisesequence identity between these chaperones and PapD ranges from 25 to56%, indicating an identical overall fold (Hung, D. L., et al., EMBO J.15: 3792-3805 (1996)).

The first studies on the mechanism of chaperone/substrate recognitionwas based on the observation that the C-termini of all known piluschaperones are extremely similar. Synthetic peptides corresponding tothe C-termini of the P-pilus proteins were shown to bind to PapD inELISA assays (Kuehn, M. J., et al, Science 262: 1234-1241 (1993)). Mostimportantly, the X-ray structures of two complexes were solved in whichPapD was co-crystallized with 19-residue peptides corresponding to theC-termini of either the adhesin PapG or the minor pilus component PapK(Kuehn, M. J., et al., Science 262: 1234-1241 (1993); Soto, G. E., etal., EMBO J. 17: 6155-6167 (1998)). Both peptides bound in an extendedconformation to a β-strand in the N-terminal chaperone domain that isoriented towards the inter-domain cleft, thereby extending a β-sheet byan additional strand. The C-terminal carboxylate groups of the peptideswere anchored via hydrogen-bonds to Arg8 and Lys112, these two residuesare invariant in the family of pilus chaperones. Mutagenesis studiesconfirmed their importance since their exchange against alanine resultedin accumulation of non-functional pilus chaperone in the periplasm(Slonim, L. N., et al., EMBO J. 11: 4747-4756 (1992)). The crystalstructure of PapD indicates that neither Arg8 nor Lys112 is involved instabilization of the chaperone, but completely solvent exposed(Holmgren, A. & Branden, C. I., Nature 342: 248-251 (1989)). On thesubstrate side the exchange of C-terminal PapA residues was reported toabolish P-pilus formation, and similar experiments on the conservedC-terminal segment of the P-pilus adhesin PapG prevented itsincorporation into the P-pilus (Hultgren, S. J., et al., “BacterialAdhesion and Their Assembly”, in: Escherichia coli and Salmonella,Neidhardt, F. C. (ed.) ASM Press, (1996) pp. 2730-2756). All evidencetherefore indicated pilus subunit recognition via the C-terminalsegments of the subunits.

A more recent study on C-terminal amino acid exchanges of the P-pilusadhesin PapG gave a more detailed picture. A range of amino acidsubstitutions at the positions −2, −4, −6, and −8 relative to theC-terminus were tolerated, but changed pilus stability (Soto, G. E., etal., EMBO J. 17: 6155-6167 (1998)).

Still, certain problems arise when this model is examined more closely.Adhesive bacterial structures not assembled to rigid, rod-like pili lackthe conserved C-terminal segments (Hultgren, S. J., et al., “BacterialAdhesion and Their Assembly”, in: Escherichia coli and Salmonella,Neidhardt, F. C. (ed.) ASM Press, (1996) pp. 2730-2756), even thoughthey are also dependent on the presence of related pilus chaperones.This indicates a different general role for the C-terminal segments ofpilus subunits, namely the mediation of quaternary interactions in themature pilus. Moreover, the attempt to solve the structure of aC-terminal peptide in complex with the chaperone by NMR was severelyhampered by the weak binding of the peptide to the chaperone (Walse, B.,et al., FEBS Lett. 412: 115-120 (1997)); whereas an essentialcontribution of the C-terminal segments for chaperone recognitionimplies relatively high affinity interactions.

An additional problem arises if the variability between the differentsubunits are taken into account. Even though the C-terminal segments areconserved, a wide range of conservative substitutions is found. Forexample, 15 out of 19 amino acid residues differ between the twopeptides co-crystallized with PapD (Soto, G. E., et al., EMBO J. 17:6155-6167 (1998)). This has been explained by the kind of interactionbetween chaperone and substrate, that occurs mainly via backboneinteractions and not specifically via side-chain interactions. Thenagain, the specificity of the chaperone for certain substrates is notreadily explained. On the contrary to the former argument, the conservedresidues have been taken as a proof for the specificity (Hultgren, S.J., et al., “Bacterial Adhesion and Their Assembly”, in: Escherichiacoli and Salmonella, Neidhardt, F. C. (ed.) ASM Press, (1996) pp.2730-2756).

The outer membrane assembly platform, also termed “usher” in theliterature, is formed by homo-oligomers of FimD or PapC, in the case ofType-1 and P-pili, respectively (Klemm, P. & Christiansen, G., Mol. Gen,Genetics 220: 334-338 (1990); Thanassi, D. G., et al., Proc. Nat. Acad.Sci. USA 95: 3146-3151 (1998)). Studies on the elongation of Type-1fimbriae by electron microscopy demonstrated an elongation of the pilusfrom the base (Lowe, M. A., et al., J. Bacteriol. 169: 157-163 (1987)).In contrast to the secretion of unfolded subunits into the periplasmicspace, the fully folded proteins have to be translocated through theouter membrane, possibly in an oligomeric form (Thanassi, D. G., et al.,Proc. Nat. Acad. Sci. USA 95: 3146-3151 (1998)). This requires first amembrane pore wide enough to allow the passage and second a transportmechanism that is thermodynamically driven (Jacob-Dubuisson, F., et al,J. Biol. Chem. 269: 12447-12455 (1994)).

FimD expression alone was shown to have a deleterious effect onbacterial growth, the co-expression of pilus subunits could restorenormal growth behavior (Klemm, P. & Christiansen, G., Mol. Gen, Genetics220: 334-338 (1990)). Based on this it can be concluded that the ushersprobably form pores that are completely filled by the pilus. Electronmicroscopy on membrane vesicles in which PapC had been incorporatedconfirmed a pore-forming structure with an inner diameter of 2 nm(Thanassi, D. G., et al., Proc. Nat. Acad. Sci. USA 95: 3146-3151(1998)). Since the inner diameter of the pore is too small to allow thepassage of a pilus rod, it has been suggested that the helicalarrangement of the mature pilus is formed at the outside of thebacterial surface. The finding that glycerol leads to unraveling of piliwhich then form a protein chain of approximately 2 nm is in goodagreement with this hypothesis, since an extended chain of subunitsmight be formed in the pore as a first step (Abraham, S. N., et al., J.Bacteriol. 174: 5145-5148 (1992); Thanassi, D. G., et al., Proc. Nat.Acad. Sci. USA 95: 3146-3151(1998)). The formation of the helical pilusrod at the outside of the bacterial membrane might then be the drivingforce responsible for translocation of the growing pilus through themembrane.

It has also been demonstrated that the usher proteins of Type-1 andP-pili form ternary complexes with chaperone/subunit complexes withdifferent affinities (Dodson, K. W., et al, Proc. Nat. Acad. Sci. USA90: 3670-3674 (1993); Saulino, E. T., et al., EMBO J. 17: 2177-2185(1998)). This was interpreted as “kinetic partitioning” that allows adefined order of pilus proteins in the pilus. Moreover, it has beensuggested that structural proteins might present a binding surface onlycompatible with one other type of pilus protein; this would be anothermechanism to achieve a highly defined order of subunits in the maturepilus (Saulino, E. T., et al., EMBO J. 17: 2177-2185 (1998)).

B. Production of Type-1 pili from Escherichia coli

E. coli strain W3110 was spread on LB (10 g/L tryptone, 5 g/L yeastextract, 5 g/L NaCl, pH 7.5, 1% agar (w/v)) plates and incubated at 37°C. overnight. A single colony was then used to inoculate 5 ml of LBstarter culture (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, pH7.5). After incubation for 24 hours under conditions that favor bacteriathat produce Type-1 pili (37° C., without agitation) 5 shaker flaskscontaining 1 liter LB were inoculated with one milliliter of the starterculture. The bacterial cultures were then incubated for additional 48 to72 hours at 37° C. without agitation. Bacteria were then harvested bycentrifugation (5000 rpm, 4° C., 10 minutes) and the resulting pelletwas resuspended in 250 milliliters of 10 mM Tris/HCl, pH 7.5. Pili weredetached from the bacteria by 5 minutes agitation in a conventionalmixer at 17.000 rpm. After centrifugation for 10 minutes at 10,000 rpmat 4° C. the pili containing supernatant was collected and 1 M MgCl2 wasadded to a final concentration of 100 mM. The solution was kept at 4° C.for 1 hour, and the precipitated pili were then pelleted bycentrifugation (10,000 rpm, 20 minutes, 4° C.). The pellet was thenresuspended in 10 mM HEPES, pH 7.5, and the pilus solution was thenclarified by a final centrifugation step to remove residual cell debris.

C. Coupling of FLAG to Purified Type-1 pili of E. coli usingm-Maleimidonbenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS)

600 μl of a 95% pure solution of bacterial Type-1 pili (2 mg/ml) wereincubated for 30 minutes at room temperature with the heterobifunctionalcross-linker sulfo-MB S (0.5 mM). Thereafter, the mixture was dialyzedovernight against 1 liter of 50 mM Phosphate buffer (pH 7.2) with 150 mMNaCl to remove free sulfo-MBS. Then 500 μl of the derivatized pili (2mg/ml) were mixed with 0.5 mM FLAG peptide (containing an amino-terminalCysteine) in the presence of 10 mM EDTA to prevent metal-catalyzedsufhydryloxidation. The non-coupled peptide was removed bysize-exclusion-chromatography.

FIG. 9 depicts an analysis of coupling of the FLAG peptide to type-1bacterial pili by SDS-PAGE. Lane 1 shows the unreacted pili subunitFimA. Lane 3 shows the purified reaction mixture of the pili with theFLAG peptide. The upper band corresponds to the coupled product, whilethe lower band corresponds to the unreached subunit.

Example 34 Construction of an Expression Plasmid for the Expression ofType-1 Pili of Escherichia coli

The DNA sequence disclosed in GenBank Accession No. U14003, the entiredisclosure of which is incorporated herein by reference, contains all ofthe Escherichia coli genes necessary for the production of type-1 pilifrom nucleotide number 233947 to nucleotide number 240543 (the fim genecluster). This part of the sequences contains the sequences for thegenes fimA, fimI, fimC, fimD, fimF, fimG, and fimH. Three different PCRswere employed for the amplification of this part of the E. coli genomeand subsequent cloning into pUC19 (GenBank Accession Nos. L09137 andX02514) as described below.

The PCR template was prepared by mixing 10 ml of a glycerol stock of theE. coli strain W3110 with 90 ml of water and boiling of the mixture for10 minutes at 95° C., subsequent centrifugation for 10 minutes at 14,000rpm in a bench top centrifuge and collection of the supernatant.

Ten ml of the supernatant were then mixed with 50 pmol of a PCR primerone and 50 pmol of a PCR primer two as defined below. Then 5 ml of a10×PCR buffer, 0.5 ml of Taq-DNA-Polymerase and water up to a total of50 ml were added. Al PCRs were carried out according to the followingscheme: 94° C. for 2 minutes, then 30 cycles of 20 seconds at 94° C., 30seconds at 55° C., and 2 minutes at 72° C. The PCR products were thenpurified by 1% agarose gel-electrophoresis.

Oligonucleotides with the following sequences with were used to amplifythe sequence from nucleotide number 233947 to nucleotide number 235863,comprising the fimA, fimI, and fimC genes: TAGATGATTACGCCAAGCTTATAATAGAAATAGTTTTTTGAAAGGAAAGCAGCATG (SEQ ID NO:196) andGTCAAAGGCCTTGTCGACGTTATTCCATTACGCCCGTC ATTTTGG (SEQ ID NO:197).

These two oligonucleotides also contained flanking sequences thatallowed for cloning of the amplification product into puc19 via therestriction sites HindIII and SalI. The resulting plasmid was termedpFIMAIC (SEQ ID NO:198).

Oligonucleotides with the following sequences with were used to amplifythe sequence from nucleotide number 235654 to nucleotide number 238666,comprising the fimD gene: AAGATCTTAAGCTAAGCTTGAATTCTC TGACGCTGATTAACC(SEQ ID NO:199) and ACGTAAAGCATTTCT AGACCGCGGATAGTAATCGTGCTATC (SEQ IDNO:200).

These two oligonucleotides also contained flanking sequences thatallowed for cloning of the amplification product into puc 19 via therestriction sites HindIII and XbaI, the resulting plasmid was termedpFIMD (SEQ ID NO:201).

Oligonucleotides with the following sequences with were used to amplifythe sequence from nucleotide number 238575 nucleotide number 240543,comprising the fimF, fimG, and fimH gene: AATTACGTGAGCAAGCTTATGAGAAACAAACCTTTTTATC (SEQ ID NO:202) and GACTAAGGCCTTTCTAGATTATTGATAAACAAAAGTCACGC (SEQ ID NO:203).

These two oligonucleotides also contained flanking sequences thatallowed for cloning of the amplification product into puc19 via therestriction sites HindIII and XbaI; the resulting plasmid was termedpFIMFGH. (SEQ ID NO:204).

The following cloning procedures were subsequently carried out togenerate a plasmid containing all the above-mentioned fim-genes: pFIMAICwas digested EcoRI and HindIII (2237-3982), pFIMD was digested EcoRI andSstII (2267-5276), pFIMFGH was digested SstII and HindIII (2327-2231).The fragments were then ligated and the resulting plasmid, containingall the fim-genes necessary for pilus formation, was termed pFIMAICDFGH(SEQ ID NO:205).

Example 35 Construction of an Expression Plasmid for Escherichia coliType-1 Pili that Lacks the Adhesion FimH

The plasmid pFIMAICDFGH (SEQ ID NO:205) was digested with Kpnl, afterwhich a fragment consisting of nucleotide numbers 8895-8509 was isolatedby 0.7% agarose gelelectrophoresis and circularized by self-ligation.The resulting plasmid was termed pFIMAICDFG (SEQ ID NO:206), lacks thefimH gene and can be used for the production of FIMH-free type-1 pili.

Example 36 Expression of Type-1 Pili Using the Plasmid pFIMAICDFGH

E. coli strain W3110 was transformed with pFIMAICDFGH (SEQ ID NO:205)and spread on LB (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, pH7.5, 1% agar (w/v)) plates containing 100 μg/ml ampicillin and incubatedat 37° C. overnight. A single colony was then used to inoculate 50 ml ofLB-glucose starter culture (10 g/L tryptone, 5 g/L yeast extract, 1%(w/v) glucose, 5 g/L NaCl, pH 7.5, 100 mg/ml ampicillin). Afterincubation for 12-16 hours at 37° C. at 150 rpm, a 5 liter shaker flaskscontaining 2 liter LB-glucose was inoculated with 20 milliliter of thestarter culture. The bacterial cultures were then incubated foradditional 24 hours at 37° C. with agitation (150 rpm). Bacteria werethen harvested by centrifugation (5000 rpm, 4° C., 10 minutes) and theresulting pellet was resuspended in 250 milliliters of 10 mM Tris/HCl,pH 8. Pili were detached from the bacteria by agitation in aconventional mixer at 17,000 rpm for 5 minutes. After centrifugation for10 minutes at 10,000 rpm, 1 hour; 4° C. the supernatant containing piliwas collected and 1 M MgCl₂ was added to a final concentration of 100mM. The solution was kept at 4° C. for 1 hour, and precipitated piliwere then pelleted by centrifugation (10,000 rpm, 20 minutes, 4° C.).The pellet was then resuspended in 10 mM HEPES, 30 mM EDTA, pH 7.5, for30 minutes at room temperature, and the pilus solution was thenclarified by a final centrifugation step to remove residual cell debris.The preparation was then dialyzed against 20 mM HEPES, pH 7.4.

Example 37 Activation of HBcAg-Lys with SPDP

HBcAg-Lys at a concentration of 15 μM was reacted with SPDP at aconcentration of 456 μM SPDP for 60 minutes at room temperature,resulting in a thirty-fold excess of cross-linker over capsid subunit.The reaction mixture was subsequently loaded on SDS-PAGE for analysis,as shown in FIG. 10. The gel shows that the monomer subunits arecross-linked to dimers and higher-order polymers during the reaction.

Example 38 Multimerization of HBcAg-Lys Upon Reaction with Sulfo-MBS

HBcAg-Lys at a concentration of 118 μM was reacted with 20 mM Sulfo-MBSfor 30 minutes at room temperature. As shown in FIG. 11, analysis of thereaction mixture by SDS-PAGE revealed that the HBcAg-Lys monomersinternally cross-linked to multimers, as reflected in the absence of aband corresponding to the subunit monomer after cross-linking.

Example 39 Conjugation of HBcAg-Lys-2cys Mut to the FLAG Peptide

HBcAg-Lys-2cys-Mut at a concentration of 80 μM was reacted with sulfa-MBS at a concentration of 8.8 mM for 30 minutes at room temperature,resulting in a 110-fold excess of cross-linker over capsid subunit. Thereaction mixture was precipitated two times with 50% ammoniumsulfate andresuspended in 20 mM Hepes, 150 mM NaCl, pH 7.4, in a volume equivalentto the reaction volume before precipitation. FLAG peptide containing anN-terminal cysteine was added at a concentration of 1.6 mM and thereaction was allowed to proceed for four hours at room temperature. Thereaction mixture was subsequently loaded on SDS-PAGE for analysis, andthe coupling products are shown in FIG. 12.

Example 40 Conjugation of Pili to the p33 Peptide

A solution of 1 ml pili at a concentration of 1.5 mg/ml (concentrationof the subunit) was reacted with 750 μl of a 100 mM Sulfo-MBS solutionin 20 mM Hepes, pH 7.4, for 45 minutes at room temperature. The reactionmixture was desalted over a Sephadex G25 column equilibrated with 20 mMHepes, pH 7.4. Fractions containing pili protein were pooled afteranalysis by dot blot stained with amidoblack, and 0.6 μl of a solutionof 100 mM p33 peptide (CGGKAVYNFATM, SEQ ID NO:175), containing anN-terminal cysteine, in DMSO was added to 100 μl of the desaltedactivated pili and reaction allowed to proceed for four hours at roomtemperature. The reaction mixture was subsequently analyzed by SDS-PAGE,as shown in FIG. 13.

Example 41 Expression of HBcAg-Lys-2cys-Mut

The plasmid coding for HBcAg-Lys-2cys-Mut was transformed into E. coliK802. A single colony was inoculated into 50 ml LB containing 100 mg/mlampicillin. The next day, the overnight culture was diluted into 2 L LBmedium containing 100 mg/ml ampicillin and grown until ID₆₀₀=0.6 at 37°C. Cells were induced with 1 mM IPTG, and grown for another 4 hours at37° C. The cells were then harvested, and the pellet resuspended in 5 mlof 10 mM Na₂HPO₄, 03 mM NaCl, 10 mM EDTA, 0.25% Tween, pH 7.0. Cellswere then disrupted by sonification, and ammoniumsulfate was added to aconcentration of 20%. The pellet was resuspended in 3 ml PBS buffer, andloaded onto a Sephacryl S-400 column. The protein peak containing thecapsid protein corresponding to the size of assembled capsid wascollected and loaded onto a hydoxyapatite column for subsequentpurification. The protein was eluted in the path through fraction.

Example 42 Coupling of DP178c Peptide, Immunization of Mice andDetermination of the IgG Subtypes

DP178c peptide is a fragment of the gp41 protein of HIV virus (Kilby, J.M. et al., Nature Medicine 4: 1302-07 (1998)); Wild, C. et al., AidsRes. Hum. Retroviruses 9: 1051-53 (1993)).

A. Coupling of DP178c to Pili

A solution of 3 ml Pili (2.5 mg/ml) produced as described in Example 33B was reacted with 500 μl of a 100 mM Sulfo-MBS solution for 45 minutesat RT. The reaction mixture was desalted on a Sephadex G25 columnequilibrated with 20 mM hepes pH 7.4, and fractions containing pili werepooled. An aliquot of 750 μul of the activated pili was diluted in 750μl DMSO, and 2-5 μl of a 100 mM DP178c solution in DMSO was added. Thereaction was left to react 4 hours at RT, and glucose was added to thereaction mixture to give a final concentration of 0.2%. This solutionwas then dialyzed against 20 mM Hepes, 0.1% glucose, pH 7.4. Thedialyzed coupled pili were centrifuged and loaded on SDS-PAGE foranalysis. The result of the coupling reaction is depicted on FIG. 14A.The sequence of the DP178c peptide (fragment of the HIV gp41 protein) isCYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID No: 176).

B. Immunization of Mice and IgG Subtype Determination

80 μg of Pili-DP178c was injected in saline intravenously into femaleBalb/c mice. These mice were boosted with the same amount of vaccine onday 14 and bled on day 24. DP178-specific IgG in serum was determined onday 24 in a DP 178 peptide specific ELISA (DP178c peptide was conjugatedto Ribonuclease A using the cross-linker SPDP). In FIG. 14B, averageresults from two mice are shown as optical densities obtained with a1:50 dilution of the serum.

Example 43 Expression and Purification of GRA2 Polypeptide

Gra2 is an antigen of Toxoplasma Gondii. The 59 c-terminal amino acidsacids of GRA2 with a c-terminal linker of 6 amino acids (GSGGCG, SEQ IDNo. 177) were cloned into the pGEX-2T vector (Pharmacia, 27-4801-01).Expression and purification of the GST-fusion protein was carried out asdescribed in the instructions. GST was cleaved from GRA2 with thrombinwhile the fusion protein was bound to glutathione-sepharose-beads andthe reaction stopped after 20 min. with 1 mM PMSF. The sepharose beadswere then pelleted by centrifugation and the supernatant containing theGRA2-polypeptide was collected. The solution was then concentrated10-fold with a Ultrafree-4 centrifugal filter-5K (Millipore, UFV4BCC25).To reduce disulfide bonds which might eventually have formed, thesolution was treated with 20 mM DTT 1 h on ice. DTT was removed byloading the protein solution on a PD10 column (Pharmacia). Proteinconcentration was determined by the Lowry test and concentration of freecysteines in an Ellmann's test. The protein was subsequently analyzed bySDS-PAGE. The GRA2 protein can however not be detected by Commassiestaining. A yield of 9 mg GRA2 was obtained from an 8 L culture. TheGRA2 amino acid sequence is KEAAGRGMVT VGKKLANVES DRSTTTTQAP DSPNGLAETEVPVEPQQRAA HVPVPDFSQGSGGCG (SEQ ID No. 178)

Example 44 Coupling of GRA2 to Pili

A. Coupling of GRA2 to Pili.

6 ml of a 2.5 mg/ml Pili protein solution (produced as described inExample 33 B) were reacted with a 50 fold molar excess of Sulfo-MBS, anddesalted over a PD10 column (Pharmacia). 1.5 ml of the reaction mixturewere loaded on one column, 1 ml was added and the first 1.5 ml werecollected. Fractions containing Pili were identified on a dot blotstained with amidoblack. A 300 μg/ml solution of GRA2 was concentrated100 fold, and 100 μl were reacted with 1.2 ml of the desalted activatedPili solution for 4 hours at RT. The reaction mixture was then dialyzedagainst 21 of a 20 mM Hepes, 150 mM NaCl, pH 7.2 overnight. FIG. 15Ashows an analysis of the coupling reaction.

B. Immunization of Mice with Pili-GRA2 and IgG Subtype Determination.

Mice, were immunized with 50 4g of Pili-GRA2 and boosted on day 14, viththe same amount of vaccine. Serum samples we're taken on day 0,6,14 and21 after the first immunization. GRA2 specific IgG in serum wasdetermined on day 21 in a GRA2 specific ELISA. Results of two individualmice in each group are shown in FIG. 15B. The titer was determined asthe dilution of sera resulting in half-maximal optical density (OD₅₀).

Example 45 Coupling of B2- and D2-Peptide to Pili

D2 and B2 peptides are sequences from the OmpC protein of Salmonellatyphi. It is an outer membrane porin. High level of antiporin antibodieshave been detected in the sera of patients with typhoid fever(Arocklasamy, A. and Krishnaswamy, S., FEBS Letters 453: 380-82 (1999)).

A. Coupling of B2- or D2-Peptides of the ompC protein of Salmonellatyphi to Pili

6 ml of a 2.5 mg/ml Pili protein solution (produced as described inExample 33 B) were reacted with a 50 fold molar excess of Sulfo-MBS, anddesalted over a PD 10 column (Pharmacia). 1.5 ml of the reaction mixturewere loaded on one column, 1 ml was added, and the first 1.5 ml werecollected. Fractions containing Pili were identified on a dot blotstained with amidoblack. An aliquot of 5 μl of a 100 mM solution ofpeptide was reacted with 2.6 ml of the desalted activated Pili solutionfor 4 hours at RT. The reaction mixture was then dialyzed against 21 ofa 20 mM Hepes, 150 mM NaCl, pH 7.2 overnight. FIG. 16A shows an analysisof the coupling reaction. The sequence of the D2 peptide is CGG TSN GSNPST SYG FAN (SEQ ID No. 179). The sequence of the B2 peptide is CGG DISNGY GAS YGD NDI (SEQ ID No. 180).

B. Immunization of Mice with Pili-B2 and IgG Subtype Determination.

Mice were immunized interaperitoneally in female Balb/c mice with 50 μgof Pili-B2 in saline and boosted on day 14 with the same amount ofvaccine, and bled on day 33. B2-peptide specific IgG in serum wasdetermined on day 33 in a B2-specific ELISA (B2 peptide was conjugatedto Ribonuclease A with the cross-linker SPDP). Average of the results oftwo individual mice are shown in FIG. 16B.

Example 46

The muTNFa peptide, comprising amino acids 22-33 of TNFα protein wascoupled to Pili as described in Example 42, except that no glucose wasadded during the final dialysis step, where the reaction solution wasdialyzed against 20 mM Hepes, pH 7.4 only. Two Balb/c female mice, 8days of age were immunized intravenously with 100 μg of Pili-muTNFαeach. These mice were boosted at day 14 with the same amount of vaccine,and bled on day 20. IgG specific for native TNFα protein in serum wasdetected at day 20 in an ELISA. As a control, preimmune sera of two micewere assayed for binding to TNFα protein. See FIG. 17. The sequence ofthe muTNFa peptide was CGGVEEQLEWLSQR (SEQ ID No. 181).

Example 47 A. Preparation of Bacterial Type-1 Pili Coupled to TNFPeptides

Two peptides comprising murine TNFα sequences were designed. Peptide3′murine TNFa II (3′-TNFaII) was SSQNSSDKPVAHVVANHGVGGC (SEQ ID No.182). Peptide 5′ murine TNFa II (5′ TNFa II) was CSSQNSSDKPVAHVVANHGV(SEQ ID No. 183). The peptides 5′-TNFa II and 3′-TNFa II were coupled tobacterial type-1 pili as follows. An aliquot of 1 ml of a Pili solution(2.5 mg/ml) was reacted with 503 μl of a 100 mM Sulfo-NMS solution for45 minutes at RT. The reaction mixture was desalted over a desaltingcolumn previously saturated with Pili protein and equilibrated in 20 mMHepes, pH 7.4. The fractions containing protein were pooled. Art aliquotof 1 ml of desalted Pili was mixed with 1.56 μl of peptide (100 mM inDMSO), and the reaction left to proceed for 4 hours at RT. The reactionsolution was then dialyzed overnight against 20 mM Hepes, 150 mM NaCl,pH 7.4 in the cold. See FIG. 18A.

B. Immunization and Detection of Antibodies Specific for native TNFα andthe 3′ TNFII and 5′ TNFII Peptides

Balb/c mice were vaccinated intraperitoneally with 30 g protein insaline, on day 0, 14 and 33. IgG antibodies specific for native TNFαprotein (FIG. 18B) and for the 3′ TNFII and 5′ TNFII peptides (FIG. 18C)were measured in a specific ELISA.

1. Native TNFα ELISA

2 μg/ml native TNFα protein was coated on ELISA plates. Sera were addedat different dilutions and bound IgG was detected with a horseradishperoxidase-conjugated anti-murine IgG antibody. Results from fourindividual mice are shown on day 21 and day 43.

2. Anti peptide ELISA

IgG antibodies specific for the 3′ TNFII and 5′ TNFII peptides weremeasured in a specific ELISA 10 ug/ml Ribonuclease A coupled to 3′ TNFIIor 5′TNFII peptide was coated on ELISA plates. Sera were added atdifferent dilutions and bound IgG was detected with a horseradishperoxidase-conjugated anti-murine IgG antibody. Results from fourindividual mice are shown on day 21.

C. Analysis of Sera From Mice Immunized Under B.: IgG SubtypeDetermination

Sera from the immunized mice described under B. were taken on day 50.Antibodies specific for the TNF peptides described under A. weremeasured in a specific ELISA on day 50. RNAse coupled to thecorresponding TNF peptide was coated on ELISA plates at a concentrationof 10 μg/ml. Sera were added at different dilutions and bound antibodywas detected with horse radish peroxidase-conjugated anti-murineantibodies. See FIG. 18D.

Example 48 Coupling of Pili to M2 Peptide, Immunization of Mice, and IgGSubtype Determination

M2 peptide was coupled to pili as described in Example 47. The peptidewas reacted at a fivefold molar excess with the activated Pili. FemaleBalb/c mice were injected with 50 μg Pili-M2 in saline subcutaneously.Mice were boosted with the same amount of vaccine on day 14 and bled onday 27, M2 specific IgG in serum was determined on day 27 in aM2-specific ELISA (peptide conjugated to Ribonuclease A with thecross-linker SPDP for coating). See FIGS. 19A and 19B.

Example 49 Immunization of Mice with HbcAg-Lys-2cys-Mut Coupled to theFlag Peptide, and IgG Subtype Determination

Flag peptide (SEQ ID NO: 147) was coupled to HBcAg-Lys-2cvs-Mut asdescribed in Example 39. Two Balb/c mice were vaccinated intravenouslywith 50 μg HBc-Ag-Lys-2cys-Mut-Flag. On day 14 mice were boosted withthe same amount of vaccine and bled on day 40, Flag-specific antibodies(Flag peptide was conjugated to Ribonuclease A with the cross-linkerSPDP for coating) in serum were measured on day 40 in a specific ELISA.ELISA plates were coated with 10 μg /ml RNAse coupled to Flag peptideand serum was added at a 1:40 dilution. Bound antibodies were detectedwith peroxidase conjugate isotype-specific IgG. Results from the twomice are shown as ELISA titers in FIG. 20.

Example 50 Purification of Type-1 Pili of Eschericia coli

Isolated Type-1 pili of Eschericia coli prepared as described in Example33B were precipitated with ammonium sulfate, added to a finalconcentration of 0.5 M, at 4° C. for 30 minutes. The pili were thenpelleted by centrifugation at 20,000 rpm for 15 min at 4° C. and thepellet was resuspended in 25 ml of 20 mM HEPES buffer, pH 7.3. Theprecipitation step was repeated once, and the final sample wasresuspended in 9 ml of 20 mM HEPES, pH 7.3 and finally dialyzed againstthe same buffer to remove residual ammonium sulfate. The pili weresubsequently purified on an SR-400 size exclusion chromatography column(20 mM HEPES, pH 7.3) and the pili containing fractions were collectedand pooled.

All patents and publications referred to herein are expresslyincorporated by reference.

The entire disclosure of U.S. application Ser. No. 09/449,631, filedNov. 30, 1999, is herein incorporated by reference. All publications andpatents mentioned hereinabove are hereby incorporated in theirentireties by reference.

1-85. (canceled)
 86. A composition comprising a bacterial pilus to whichan antigen or antigenic determinant has been attached by a covalentbond.
 87. The composition of claim 86, wherein said bacterial pilus is aType 1 pilus of Escherichia coli.
 88. The composition of claim 86,wherein the pilin subunit of said Type 1 pilus comprises the amino acidsequence shown in SEQ ID NO:146 or a sequence having at least 65, 70,75, 80, 85, 90 or 95% sequence identity to SEQ ID NO:146.
 89. Thecomposition of claim 86, wherein said antigen is selected from the groupconsisting of: (a) an antigen suited to induce an immune responseagainst bacteria, (b) an antigen suited to induce an immune responseagainst viruses, (c) an antigen suited to induce an immune responseagainst parasites, (d) an antigen suited to induce an immune responseagainst cancer cells, (e) an antigen suited to induce an immune responseagainst allergens, (f) an antigen suited to induce an immune response ina farm animals, and (g) a protein suited to induce an immune response ina pet.
 90. A method of immunizing, comprising administering to a subjectthe composition of claim
 86. 91. A method of making the composition ofclaim 86, comprising combining said pilus and said antigen or antigenicdeterminant, wherein said pilus and said antigen or antigenicdeterminant interact to form an antigen array.
 92. A compositioncomprising: (a) a non-natural molecular scaffold comprising: (i) a coreparticle selected from the group consisting of: (1) a bacterial pilus orpilin protein; and (2) a recombinant form of a bacterial pilus or pilinprotein; and (ii) an organizer comprising at least one first attachmentsite, wherein said organizer is connected to said core particle by atleast one covalent bond; and (b) an antigen or antigenic determinantwith at least one second attachment site, said second attachment sitebeing selected from the group consisting of: (i) an attachment site notnaturally occurring with said antigen or antigenic determinant; and (ii)an attachment site naturally occurring with said antigen or antigenicdeterminant, wherein said second attachment site is capable ofassociation through at least one non-peptide bond to said firstattachment site; and wherein said antigen or antigenic determinant andsaid scaffold interact through said association to form an ordered andrepetitive antigen array.
 93. The composition of claim 92, wherein saidbacterial pilus is a Type-1 pilus of Eschericia coli.
 94. Thecomposition of claim 92, wherein the pilus subunit of said type-1 piluscomprises the amino acid sequence of SEQ ID NO:146 or a sequence havingat least 65, 70, 75, 80, 85, 90 or 95% sequence identity to SEQ IDNO:146.
 95. The composition of claim 92, wherein said first and/or saidsecond attachment sites comprise: (a) an antigen and an antibody orantibody fragment thereto; (b) biotin and avidin; (c) strepavidin andbiotin; (d) a receptor and its ligand; (e) a ligand-binding protein andits ligand; (f) interacting leucine zipper polypeptides; (g) an aminogroup and a chemical group reactive thereto; (h) a carboxyl group and achemical group reactive thereto; (i) a sulfhydryl group and a chemicalgroup reactive thereto; or (j) a combination thereof.
 96. Thecomposition of claim 92, wherein said first attachment site is an aminogroup and said second attachment site is a sulfhydryl group.
 97. Thecomposition of claim 92, wherein said antigen is selected from the groupconsisting of: (a) an antigen suited to induce an immune responseagainst bacteria; (b) an antigen suited to induce an immune responseagainst viruses; (c) an antigen suited to induce an immune responseagainst parasites; (d) an antigen suited to induce an immune responseagainst cancer cells; (e) an antigen suited to induce an immune responseagainst allergens; (f) an antigen suited to induce an immune response ina farm animal; and (g) a protein suited to induce an immune response ina pet.
 98. A method of immunizing, comprising administering to a subjectthe composition of claim
 92. 99. A composition comprising: (a) anon-natural molecular scaffold comprising: (i) a Hepatitis B viruscapsid protein, modified such that the cysteine residues correspondingto positions 48 and 107 in SEQ ID NO:134 are either deleted orsubstituted with another amino acid; and (ii) an organizer comprising atleast one first attachment site, wherein said organizer is connected tosaid core particle by at least one covalent bond; and (b) an antigen orantigenic determinant with at least one second attachment site, saidsecond attachment site being selected from the group consisting of: (i)an attachment site not naturally occurring with said antigen orantigenic determinant; and (ii) an attachment site naturally occurringwith said antigen or antigenic determinant, wherein said secondattachment site is capable of association through at least onenon-peptide bond to said first attachment site; and wherein said antigenor antigenic determinant and said scaffold interact through saidassociation to form an ordered and repetitive antigen array.
 100. Thecomposition of claim 99, wherein said Hepatitis B virus capsid proteincomprises an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of SEQ ID NO:89; (b) the amino acid sequenceof SEQ ID NO:90; (c) the amino acid sequence of SEQ ID NO:93; (d) theamino acid sequence of SEQ ID NO:98; (e) the amino acid sequence of SEQID NO:99; (f) the amino acid sequence of SEQ ID NO:102; (g) the aminoacid sequence of SEQ ID NO:104; (h) the amino acid sequence of SEQ IDNO:105; (i) the amino acid sequence of SEQ ID NO:106; (j) the amino acidsequence of SEQ ID NO:119; (k) the amino acid sequence of SEQ ID NO:120;(l) the amino acid sequence of SEQ ID NO:123; (m) the amino acidsequence of SEQ ID NO:125; (n) the amino acid sequence of SEQ ID NO:131;(o) the amino acid sequence of SEQ ID NO:132; (p) the amino acidsequence of SEQ ID NO:134; (q) the amino acid sequence of SEQ ID NO:157;and (r) the amino acid sequence having at least 90% identity to any oneof the above (a) to (p).
 101. The composition of claim 99, wherein saidfirst and/or said second attachment sites comprise: (a) an antigen andan antibody or antibody fragment thereto; (b) biotin and avidin; (c)strepavidin and biotin; (d) a receptor and its ligand; (e) aligand-binding protein and its ligand; (f) interacting leucine zipperpolypeptides; (g) an amino group and a chemical group reactive thereto;(h) a carboxyl group and a chemical group reactive thereto; (i) asulfhydryl group and a chemical group reactive thereto; or (j) acombination thereof.
 102. The composition of claim 99, wherein saidorganizer is a polypeptide or residue thereof, wherein said secondattachment site is a polypeptide or residue thereof, and wherein firstattachment site is a lysine residue and said second attachment site is acysteine residue.
 103. The composition of claim 99, wherein said antigenis selected from the group consisting of: (a) an antigen suited toinduce an immune response against bacteria, (b) an antigen suited toinduce an immune response against viruses, (c) an antigen suited toinduce an immune response against parasites, (d) an antigen suited toinduce an immune response against cancer cells, (e) an antigen suited toinduce an immune response against allergens, (f) an antigen suited toinduce an immune response in a farm animals, and (g) a protein suited toinduce an immune response in a pet.
 104. A method of immunizing,comprising administering to a subject the composition of claim
 99. 105.A method of making the composition of claim 99, comprising combiningsaid non-natural molecular scaffold and said antigen or antigenicdeterminant, wherein said non-natural molecular scaffold and saidantigen or antigenic determinant interact to form an antigen array.